TECOmedical Clinical & Technical Review

Measurement of
Complement Activation
in Human Disease

Authors:
Peter Haima, Ph.D.
Life-Force biomedical communication, Netherlands
CONTENTS
1 COMPLEMENT SYSTEM 3
1.1 The complement cascade 3
1.2 Activation of the complement system 4
Activation of the Classical Pathway 4
Activation of the Lectin Pathway 4
Activation of the Alternative Pathway 4
C3 Cleavage 4
Activation of the Terminal Pathway 5
Regulation after initiation of the complement cascade 5
1.3 Adverse Effects Of The Complement System 5
Hypersensitivity or infusion reactions 5
Anaphylatoxins, inflammation and cancer 5

2 COMPLEMENT IN HUMAN DISEASE 6

2.1 Measurement of complement Activation in human disease 7
Systemic Lupus Erythematosus 8
Age related macular degradation 9
Hereditary angioedema (HAE) 11
Ischemia/reperfusion(I/R)injury 12
Rheumatoid arthritis 13
Hemolytic Uremic Syndrome 14
Paroxysmal Nocturnal Hemoglobinuria 16
Membranoproliferative Glomerulonephritis 17
Alzheimer’s Disease 18
Cancer 19

3 OTHER DISEASES 20
3.1 Lyme Disease 20
3.2 Allergic Asthma 20

4 COMPLEMENT ELISA’S 21
4.1 Overview of available complement ELISA’s 21
4.2 Technical information on complement ELISA’s 21
4.3 Cross reactivity of complement ELISA’s to primate samples 33

5 COMPLEMENT ANTIBODIES AND REAGENTS 35

5.1 Product information monoclonal antibodies 38
5.2 Animal species crossreactivity monoclonal antibodies 41
5.3 Animal species crossreactivity polyclonal antibodies 42

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 1 COMPLEMENT SYSTEM
The complement system (C-system) helps or “complements” the ability of antibodies and phagocytic cells to clear
invading cellular pathogens (e.g. bacteria) [2, 3] by orchestrating their clearance by macrophages and recognition
by lymphocytes for specific long-term immunity. It is part of the immune system called the innate immune system
that is not adaptable and does not change over the course of an individual’s lifetime. The complement system con-
sists of proteins and glycoproteins found in the blood, generally synthesized by the liver, and normally circulating as
inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific
proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end-result of this activation
cascade is massive amplification of the response, a massive acute inflammatory reaction and activation of the cell-
killing membrane attack complex (MAC). As revealed recently, the C system also has important activities in some non-
immune physiological processes, such as conception and tissue regeneration.

THE COMPLEMENT CASCADE

Figure 1, The complement cascade (Figure was adapted from Rutkowski et al. (2010) [7].) The complement cascade comprises the classic, alternative, and
MBL pathways. The classic pathway is activated by the Fc portion of immunoglobulins bound to antigen, apoptotic cells, Gram-negative bacteria, and
viruses. The C1 complex, made up of C1q, C1r, and C1s subunits, initiates the downstream classic cascade. Upon binding of C1q to an inciting stimulus, C1r
catalyzes breakage of a C1s ester bond, resulting in its activation and subsequent cleavage of C2 and C4 into their respective “a” and “b” fragments. The
formation of C2a4b creates C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to other C3 convertases, forming C2a4b3b, also known as C5
convertase. It facilitates the final steps of the cascade by splitting C5 into C5a and C5b. The latter fragment is the critical first protein that combines with C6,
C7, C8, and multiple C9 proteins to form the MAC, the terminal, pore-forming complement protein complex responsible for lysis of cells and pathogens. The
MBL pathway is activated by surfaces bearing mannose groups or other pathogen-associated molecular patterns. MBL or ficolin activation of mannose-
associated serine proteases (MASP) results in cleavage of C2 and C4 similar to the C1 complex, with subsequent production of C3 convertase and comple-
ment cascade activation resembling the classic pathway. Lastly, the alternative pathway is activated by a multitude of infectious agents including various
bacteria, viruses, and fungi, as well as neoplastic cells. This pathway exhibits a unique “tickover” effect whereby low-level C3 cleavage occurs continuously.
Generated C3b binds Bb, a cleavage fragment of factor B, and properdin, resulting in the formation of the alternative pathway C3 convertase. Binding of
additional C3b to the alternative pathway C3 convertase renders it capable of C5 cleavage, and forms the basis for the amplification loop of the alterna-
tive pathway. Additionally, C3b generated by alternative pathway C3 convertase can attach to target surfaces and bind Bb, forming a C3 convertase that
amplifies downstream complement proteins locally at the target surface. Although the activation and amplification of the three pathways differ initially,
they commonly cleave C3 into C3a and C3b, finally resulting in terminal formation of the MAC.

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1.2 ACTIVATION OF THE COMPLEMENT SYSTEM

The three pathways of activation all generate homologous variants of the protease C3-convertase. The classical com-
plement pathway (C1, C4, C2) is activated by the presence of antigen-antibody immune complexes and some viral
envelopes. The Lectin and Alternative pathways can be activated by C3 hydrolysis or antigens without the presence
of antibodies (non-specific immune response, see Figure 1 for details). With few exceptions, if there is no cleavage of
protein C3, there is no significant activation of the Complement System. C3 is the most abundant complement pro-
tein in the blood (> 1000 μg/mL).

ACTIVATION OF THE CLASSICAL PATHWAY

The classic pathway is activated by the Fc portion of immunoglobulins bound to antigen, apoptotic cells, Gram-ne-
gative bacteria, and viruses. The C1 complex, made up of C1q, C1r, and C1s subunits, initiates the downstream clas-
sic cascade. Upon binding of C1q to an inciting stimulus, C1r catalyzes breakage of a C1s ester bond, resulting in its
activation and subsequent cleavage of C2 and C4 into their respective “a” and “b” fragments. The formation of C2a4b
creates C3 convertase, which cleaves C3 into C3a and C3b.

ACTIVATION OF THE LECTIN PATHWAY

The MBL or Lectin pathway is activated by surfaces bearing mannose groups or other pathogen-associated molecular
patterns. MBL or ficolin activation of mannose-associated serine proteases (MASP-1, -2 and -3) results in cleavage of
C2 and C4 similar to the C1 complex, with subsequent production of C3 convertase (C2a4b) and complement cascade
activation resembling the classic pathway [4]. To prevent excessive Complement activation in vivo, Factor I (another
Complement protein) cleaves C4b into two new fragments: C4d and C4c. This inactivates the C3 convertase.

Since C4d is unique to the classical and lectin pathway, detection of C4d in a test sample is confirmation of C4
activation and ergo, classical or lectin pathway activation [5].

ACTIVATION OF THE ALTERNATIVE PATHWAY

The alternative pathway is activated by a multitude of infectious agents including various bacteria, viruses, and fungi,
as well as neoplastic cells. This pathway exhibits a unique “tickover” effect whereby low-level C3 cleavage occurs
continuously. Generated C3b binds Bb, a cleavage fragment of factor B, and properdin, resulting in the formation of
the alternative pathway C3 convertase. Binding of additional C3b to the alternative pathway C3 convertase renders
it capable of C5 cleavage, and forms the basis for the amplification loop of the alternative pathway. Additionally, C3b
generated by alternative pathway C3 convertase can attach to target surfaces and bind Bb, forming a C3 convertase
that amplifies downstream complement proteins locally at the target surface.

Since Factor B is unique to the alternative pathway, detection of Bb in a test sample confirms activation of
Factor B and so, the alternative pathway.

C3 CLEAVAGE
Once either the classical, lectin, or alternative pathway is activated, the C3 convertase (C2a4b) is formed. This enzyme
cleaves C3 into C3b and C3a (a potent anaphylatoxin). C3b is responsible for many of the biological activities of com-
plement. It can deposit covalently on nearby surfaces containing a free amino or hydroxyl group (such as on bacteria),
which leads to opsonization and ultimately phagocytosis. It can, via a feedback loop, generate more alternative pa-
thway C3 convertase. It can also participate in the formation of C5 convertase. C3b is controlled in vivo by proteolysis
of C3b to iC3b (I = inactivated). The proteolysis is accomplished by Factor I in the presence of cofactors (Factor H or
Complement Receptor 1, CR1). The reaction can occur either in the fluid phase or with C3b that is covalently bound
to cell surfaces.

Because C3 can be activated by either the classical, lectin or alternative pathway, detection of either iC3b or
C3a in a test specimen is proof of Complement activation by one or both of these pathways. Increases in either
fragment in human serum exposed to a biomaterial (when in a controlled environment) is proof that the bio-
material activates the Complement System.

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 ACTIVATION OF THE TERMINAL PATHWAY
C3b generated by any of the three pathways binds to other C3 convertases, forming C2a4b3b, also known as C5
convertase. This enzyme cleaves C5 into C5a (the most potent anaphylatoxin) and C5b. C5b interacts with four other
Complement proteins, C6, C7, C8 and C9, to form the Membrane Attack Complex (MAC or Terminal Complement
Complex, TCC) resulting in cell lysis. In in vitro test samples, much of the C5b generated does not result in cell lysis, but
is diverted to the fluid phase to form a soluble, lytically inactive complex – SC5b-9.

Because C5 is unique to the terminal pathway, detection of C5a or SC5b-9 in a test sample is confirmation of
terminal pathway activation in the specimen.

REGULATION AFTER INITIATION OF THE COMPLEMENT CASCADE

After initiation of the complement cascade, the activation is non-specific, i.e. molecules can react on any surface
which is nearby. So tight regulation by the host is crucial. Several secreted regulators like C1-inhibitor (which inacti-
vates the C1 complex), C4 binding protein (C4BP) factor I, and factor H, accelerate the dissociation of the two C3 con-
vertases and inactivate the C4b and C3b molecules. To prevent activation of complement on host tissue, membrane
bound complement regulators, such as MCP (Membrane co-factor protein, CD46), DAF (decay accelerating factor,
CD55) and Protectin (CD59), can either accelerate the dissociation of the C3-convertases or prevent integration of the
C5b-9 complex.

1.3 ADVERSE EFFECTS OF THE COMPLEMENT SYSTEM

HYPERSENSITIVITY OR INFUSION REACTIONS

One of the illnesses where abnormal C activation plays a causal role is a hypersensitivity syndrome known as infusion-
or anaphylactoid reaction caused by medical devices, i.v. medicines or diagnostic agents. Non-IgE-mediated anaphy-
lactoid, or pseudoallergic reactions may represent as high as 77% of all immune-mediated drug reactions implying
hundreds of thousands of reactions each year, with death occurring in a small, but not insignificant percentage [8].

ANAPHYLATOXINS, INFLAMMATION AND CANCER

The complement cascade contains some of the most powerful proinflammatory molecules in the body, including
most notably the anaphylatoxins C3a C4a and C5a [9,10]. They cause smooth muscle contraction, histamine release
from mast cells, and enhanced vascular permeability [11, 12]. They also mediate chemotaxis, inflammation, and ge-
neration of cytotoxic oxygen radicals [11]. The contribution of the complement cascade to acute inflammation is well
established, as is the continuous activation and consumption of complement proteins in chronic inflammatory states
[9, 10]. This latter property is a recognized pathogenic factor in a wide spectrum of chronic inflammatory diseases,
including rheumatoid arthritis (13), glomerulonephritis (14), atherosclerosis (15), asthma (16, 17), and multiple sclero-
sis (18). Evidence is accumulating that complement proteins may also facilitate the major features of carcinogenesis,
including dysregulation of mitogenic signaling pathways, sustained cellular proliferation, angiogenesis, insensitivity
to apoptosis, invasion and metastasis, and escape from immunosurveillance [7].

REFERENCES
[1] Liszewicki MK and Atkinson JP. The Complement System.
In: Fundamental Immunology. Paul, WE (ed.) (1993).
[2] Botto M et al. Complement in human diseases: Lessons from
complement deficiencies. Mol. Immunol. 46(4):2774-2783 (2009).
[3] Wallis R et al. Paths reunited: initiation of the classical and
lectin pathways of complement activation. Immunobiol. 215(1):
1-11 (2010).
[4] Degn S et al. Map44, a human protein associated with
pattern recognition molecules of the complement system and
regulating the lectin pathway of complement activation.
J. Immunol. 183(11):7371-7378 (2009).
[5] American Society for Testing and Materials. F2567-06: Standard
Practice for Testing For Classical Pathway Complement Activation
in Serum by Solid Materials. (2010).
[6] American Society for Testing and Materials. F2065-00: Standard
Practice for Testing For Alternative Pathway Complement
Activation in Serum by Solid Materials. (2010).
[7] Rutkowski MJ, Sughrue ME, Kane AJ, et al. Cancer and the
Complement Cascade. Mol Cancer Res 8:1453-1465 (2010).
[8] Szebeni J. Hemocompatibility testing for nanomedicines
and biological: predictive assays for complement mediated
infusion reactions. Eur. J. Nanomed. 4(1):33-53 (2012).

5 TECOmedical
[9] Guo RF, Ward PA. Role of C5a in inflammatory responses. [14] Welch TR. Complement in glomerulonephritis. Nat Genet
Annu Rev Immunol 23:821–52 (2005). 31:333–4 (2002).

[10] Kohl J. Anaphylatoxins and infectious and non-infectious [15] Niculescu F, Rus H. The role of complement activation in
inflammatory diseases. Mol Immunol 38:175–87 (2001). atherosclerosis. Immunol Res 30:73–80 (2004).

[11] Gennaro R et al. C5a fragment of bovine complement. [16] Hawlisch H et al. The anaphylatoxins bridge innate and
Purification, bioassays, amino-acid sequence and other structural adaptive immune responses in allergic asthma. Mol Immunol
studies. Eur. J. Biochem. 155 (1): 77–86 (1986). 41:123–31 (2004).

[12] Rosa PA et al. Sequence of the gene for murine complement [17] Humbles AA et al. A role for the C3a anaphylatoxin receptor
component C4. J. Biol. Chem. 264 (28): 16565–16572. (1989). in the effector phase of asthma. Nature 406: 998–1001 (2000).

[13] Linton SM et al. Complement activation and inhibition in [18] Storch MK et al. Multiple sclerosis: in situ evidence for an-
experimental models of arthritis. Mol Immunol 36:905–14 (1999). tibody- and complement-mediated demyelination. Ann Neurol
43:465–71 (1998).

2 COMPLEMENT IN HUMAN DISEASE

Complement deficiencies form about 2% of all primary immunodeficiency disorders [1]. Deficiencies in the comple-
ment cascade can lead to immunocompromise and overwhelming infection and sepsis. In addition to playing an
important role in host defense against infection, the complement system is a mediator in both the pathogenesis and
prevention of immune complex diseases, such as systemic lupus erythematosus (SLE). These findings underscore the
duality of the complement system. It has a protective effect when functioning in moderation against pathogens; at
the same time, the inflammation promoted by complement activation can result in cellular damage when not kept
in check.
Investigation of possible complement deficiencies focus on:
• Clarification of complement deficiencies
• Detection of activation of the complement system
• Verification of adequate regulation of the complement system

Complement defects can be primary (hereditary) or secondary (acquired). Hereditary defects make up approx. 10 % of
all primary humoral immune deficiencies and are typically recessive autosomal in origin. Consequently, heterozygous
carriers usually remain asymptomatic. Secondary deficiencies are caused by complement consumption, reduced syn-
thesis and/ or increased catabolism. A lack of components of the classical activation sequence (C1, C4, C2) is often
associated with autoimmune processes (SLE and clinical pictures similar to lupus). Recurrent bacterial infections are
indicative of defects of the components C3 – C8. Infections with Neisseria predominate in the case of deficiency of
the late components. Defects are described for nearly all complement and regulator proteins with the exception of
factor B (see figures 2, and 3) [1-5].

Increased and decreased values of the different complement factors or their metabolites are used for
diagnostic purposes:

6
 Figure 3. Disease associated
with complement factors.

2.1 MEASUREMENT OF COMPLEMENT ACTIVATION IN

HUMAN DISEASE

Monitoring the development of complement activation is necessary to detect patients at risk after polytrauma, burns,
sepsis, transplantation and other complement-related disease (see next chapter for details). Repeated monitoring of
activation products helps to assess immunological-inflammatory activity, especially in the control of immune sup-
pressive therapy. All components and regulators should be included in a comprehensive complement diagnostics;
this is true both with respect to their plasma concentration and their function within the cascade reaction. Modern
complement diagnostics, which safely identifies a defect or an activation of the system, requires a step-by-step appro-
ach. For an initial examination, we recommend assessing the total haemolysis activity of the classical pathway (CH50),
the alternative pathway (APH50) and the detection of the pathway-independent complement activation product
C3d (Figure 4). A complete overview of analytical methods available for complement diagnostics is shown in Figure 5.

Figure 4. Parameters for initial

examination of complement ac-
tivation.

Figure 5. Analytical methods for

complement diagnostics.

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REFERENCES

[1] Cooper MA et al. Primary immunodeficiencies. Am Fam Phy-
sician. 68(10):2001-8 (2203).
[2] Chaganti RK et al. Complement Deficiencies. eMedicine,
Jul 2009.
[3] Agrawal R et al. Complement Deficiency. eMedicine,
May 2009.
[4] Jonsson G et al. Rheumatological manifestations, organ da-
mage and autoimmunity in hereditary C2 deficiency. Rheumato-
logy (Oxford). 46(7):1133-9. Epub (2007).
[5] Jonsson G et al. Hereditary C2 deficiency in Sweden: fre-
quent occurrence of invasive infection, atherosclerosis, and rheu-
matic disease. Medicine (Baltimore) 84(1):23-34 (2005).

SYSTEMIC LUPUS ERYTHEMATOSUS

Systemic Lupus Erythematosus (SLE, lupus) is an autoimmune disease in which the immune system attacks the body’s
cells and tissues, resulting in inflammation and tissue damage. SLE is a Type III hypersensitivity in which antibody-
immune complexes precipitate and cause a further immune response. Periods of acute illness (called flares) alternate
with periods of remission. SLE most often affects the heart, joints, skin, lungs, blood vessels, liver, kidneys and nervous
system. It occurs nine times more frequently in women than men, usually during the childbearing years (ages 15-35)
[1]. The American College of Rheumatologists (ACR) established eleven criteria [2,3] to identify patients with SLE for
clinical studies. A person has SLE if any 4 out of the 11 symptoms are present simultaneously or serially on two sepa-
rate occasions. One important symptom is blood – hematologic disorder – hemolytic anemia or leukopenia, lymph-
openia or thrombocytopenia in the absence of offending drug.

Hypocomplementemia is also seen, due to either consumption of C3 and C4 by immune complex-induced in-
flammation or to congenital complement deficiency (which may predispose to SLE).

The pathogenic mechanisms in SLE are complex and there are several hypotheses concerning these mechanisms,
based on the relationship between complement and the disease. One of the first was that impaired handling of im-
mune complexes was a major factor in SLE. Apoptotic cells are considered to be the main source of auto-antigen and
the C1q protein, along with others (such as C-reactive protein (CRP) and IgM) are responsible for recognition. Low
concentrations of complement components are found in a majority of patients with active and severe SLE [4]. Defici-
encies in proteins involved in the classical complement pathway, such as C1, C2 and C4, are associated with active SLE
[5,6]. Decreases in C1q levels have been noted prior to the clinical manifestations of SLE flares. Several autoantibodies
which have a high affinity for complement proteins have been identified in the sera of patients with SLE. One of the
first was an antibody against and activation fragment of C3, iC3b. The precise role of this autoantibody in this disease
(if any) has yet to be determined. Other, more rare, antibodies against C3 nephritic factor, C1 Inhibitor and the C4b2a
(C3 Convertase of the Classical pathway) have also been identified in a small number of patients. The most prevalent
autoantibody is anti-C1q, which is found in approximately one-third of SLE patients, especially those with severe
disease. There is an association between the presence of the anti-C1q autoantibodies and glomerulonephritis. There
are usually indications of intense activation of the classical pathway of complement (demonstrated by low C1q and

8
 C4 levels). Levels of C3, which are usually well maintained in patients with SLE, may be substantially below normal
levels in those patients with anti-C1q autoantibodies [7].

Conversely, increases in the levels of the anaphylatoxins, C5a and C3a, and deposition of the Membrane Attack Com-
plex (MAC) in the kidneys and lungs have been reported and are related to tissue damage. Low levels of Complement
Receptor type I (CR1) and deposition of C4 and C3 fragment on erythrocytes in active SLE have been made by several
researchers. It has also been demonstrated that the combination of high levels of erythrocyte-bound C4d and low
levels of CR1 had high sensitivity (72%) and specificity (79%) for SLE [8].

Treatment of SLE by manipulation of the complement system is very complex. Complement deficiencies are a signifi-
cant cause of SLE. So, it would seem that increasing complement activity by replacement of deficient proteins would
be appropriate. Unfortunately, there are no sources of purified or recombinant complement proteins that have been
approved for therapeutic purposes. In addition, increased activation of complement could result in patients with an
accumulation of immune complexes could result in inflammatory injury. Finally, it is possible that the introduction of
these “foreign” proteins could induce production of antibodies which would reduce or eliminate the effectiveness of
the treatment.

Further complicating matters, there is ample evidence that complement components, specifically the anaphylatoxins
and the MAC, cause tissue damage associated with SLE. This would indicate that inhibition of the complement system
is the appropriate treatment. This approach is being studied in the treatment of other diseases caused by comple-
ment activation, such as glomerulonephritis, ischemia reperfusion injury and xenograft rejection. Studies have shown
that anti-C5a antibodies have been therapeutically effective in these conditions. Trials using these antibodies have
been proposed for patients suffering from SLE [9].

REFERENCES

[1] Rahman A and Isenberg DA. Review article: Systemic Lupus [6] Sturfelt G and Truedsson L. Complement and its breakdown
Erythematosus. N. Engl. J. Med. 358(9):929-939 (2008). products in SLE. Rheum. 44:1227-1232 (2005).

[2] Tan EM et al. The 1982 revised criteria for the classification
of systemic lupus erythematosus. Arthritis Rheum. 25:1271-1277
(1982).
[3] Hochberg MC. Updating the American College of Rheuma-
tology revised criteria for the classification of systemic lupus ery-
thematosus [letter]. Arthritis Rheum. 40:1725 (1997).
[4] Schur PH and Sandsson J. Immunological factors and clinical
activity in systemic lupus erythematosus. N. Engl. J. Med. 278:533-
538 (1986).
[7] Walport MJ. Supplement Review: Complement and systemic
lupus erythematosus. Arthritis Res. 4(suppl 3):S279-S293 (2002).
[8] Teixeira JE at al. CR1 stump peptide and terminal comple-
ment complexes are found in the glomeruli of lupus nephritis
patients. Clin. Exp. Immunol. 105:497-503 (1996).
[9] Holers VM. Complement as a regulatory and effector
pathway in human disease. In: Therapeutic interventions in the
complement system. Lambris JD, Holers, VM (eds). pgs. 1-32 (2000).

[5] Taylor, PR et al. A hierarchical role for classical pathway comple-

ment proteins in the clearance of apoptotic cells in vivo. J. Exp. Med.
192:359-366 (2000).

AGE RELATED MACULAR DEGRADATION

Age-related Macular Degeneration (AMD) affects an estimated 30-50 million people worldwide and is the leading
cause of blindness in the elderly population, particularly those of European descent. Early disease is characterized by
the deposition of lipoproteinaceous deposits (called drusen) which accumulate in the space between Bruch’s mem-
brane and the retinal pigment epithelium (RPE). Progression of the disease results in two severe forms: geographic
atrophy (GA, also referred to as the “dry” form of the disease) characterized by loss of RPE and outer neurosensory
retinal cells, and chroidal neovascularization (CNV, also referred to as the “wet” form of the disease) characterized by
the growth of blood vessels into the retinal layer which leak fluid or bleed [1, 2]. The World Health Organization (WHO)
reported in 2002 that 8.7% of the world’s blindness was related to AMD, with 14 million persons worldwide blind or
severely visually impaired by AMD.

9 TECOmedical
Studies on the composition of drusen have implicated inflammation, specifically local activation of the alternative
complement pathway (AP), in the pathogenesis of AMD [3]. C3 complement fragments, C5 and the membrane attack
complex (MAC, C5b-9) have been identified in drusen, sub-RPE space and within the capillary pillars of the choroid. In
addition fluid phase regulatory proteins, such as clusterin and vitronectin have also been observed in drusen [4]. Ad-
ditional research has shown that variants in the complement Factor H (CFH) gene are significantly associated with an
increased risk for developing AMD [5-7]. Factor H is synthesized predominantly in the liver, but there is also local syn-
thesis in the RPE cells. [8]. Factor H is the major inhibitor of AP in the fluid phase. It binds to the host cell and inhibits
activation by interfering with the formation and activity of the alternative C3 convertase (C3bBb). CFH acts as a cofac-
tor for the inactivation of C3b by Factor I. In the absence of CFH, C3b binds Factor B, allowing its cleavage into Ba and
Bb fragments by Factor D. This eventually results in the formation of the alternative C5 convertase and subsequent
assembly of terminal complement pathway components into the MAC. Small differences in the concentration or ac-
tivity of regulatory proteins (Factor H, Factor I or Factor D) could have an impact on the extent of local complement
activation within the eye. Therefore, low level activation of the AP could result in the local release of pro-inflammatory
and angiogenic compounds and also tissue damage in the retina – leading to manifestation of the disease [9].

A number of AMD therapies are in development which target specific components of the complement system (Figure
6). These therapies have the potential to work at earlier stages of the disease, preventing the progression to the later
phases of AMD. However, any therapeutic method must minimize the systemic effects of complement inhibition on
the immune system. The use of monoclonal antibodies and nucleic acid aptamers have both been proposed for ma-
nagement of AMD.

Figure 6. Current medications in development for the management of dry AMD [10].

REFERENCES

[1] De Jong PT. Age-related macular degeneration. N. Engl. J.
Med. 255(14):1474-1485 (2006).
[2] Congdon N et al. Causes and prevalence of visual im-
pairment among adults in the United States. Arch. Ophthalmol.
122:477-485 (2004).
[3] Hageman GS et al. An integrated hypothesis that considers
drusen as biomarkers of immune-mediated processes at the RPE-
Bruch’s membrane interface in aging and age-related macular
degeneration. Prog. Retin. Eye Res. 20:705-732 (2001).
[6] Ufret-Vincenty RL et al. Transgenic mice expressing variants
of complement factor H develop AMD-like retinal findings. Inv.
Ophthal. & Vis. Sci. 51(11):5878-5887 (2010).
[7] Zipfel PF et al. The role of complement in AMD. In: Inflamma-
tion and Retinal Disease: Complement Biology and Pathology.
Lambris JD, Adamis, AP (eds). Advances in Experimental Medicine and
Biology, pgs. 9-24 (2010).
[8] Klein RJ et al. Complement factor H polymorphism in age-
related macular degeneration. Science 308:385-389 (2005).

[4] Sivaprasad S and Chong, NV. The complement system and [9] Scholl HPN et al. Systemic complement activation in age-
age-related macular degeneration. Eye 20:867-872 (2006). related macular degeneration. PLoS ONE 3(7):e2593-e2599 (2008).

[5] okiranta TS et al. Analysis of the recognition mechanism [10] Punjabi OS and Kaiser PK. Dry AMD: The role of complement.
of the alternative pathway of complement by monoclonal anti- Rev. Ophthal. 19(10):64-68 (2012).
factor H antibodies: Evidence for multiple interactions between
H and surface bound C3b. FEBS Letts. 393:297-302 (1996).

10
 HEREDITARY ANGIOEDEMA (HAE)
Hereditary Angioedema (HAE) was first described by Heinrich Quincke in the 1850’s and again in the 1880’s by Sir
William Osler. The rare, inherited blood disorder is characterized by episodic attacks of swelling (edema) that may
affect the face, extremities, genitals, gastrointestinal tract and upper airways. Abdominal swelling involves pain, vo-
mitingand diarrhea. Swelling of the airway can be fatal. Landerman found that serum from a patient with HAE lacked
inhibitory activity for several components of the clotting system.1 In 1963, Donaldson and Evans observed that C1 In-
hibitor was deficient in serum from HAE patients.2 Thus, C1 Inhibitor seems to possess inhibitory protein interactions
with components of clotting (such as Factor XII, kallikrein, and bradykinin). HAE is the only complement deficiency
that is transmitted as a dominant trait (i.e. a single abnormal gene in combination with a normal gene can produce
the deficiency) [3].

Hereditary Angioedema Type I (HAE-I)

Constitutes 80-85% of all HAE patients. C1 Inhibitor levels are considerably below normal due to a defective gene on
Chromosome 11. This may be due to heredity, but there are also a number of cases resulting from spontaneous mu-
tation of the gene. In addition, complement protein C4 is almost always low, while C3 and C1q are normal. HAE-I does
not respond to antihistamines or corticosteroids.

Hereditary Angioedema Type II (HAE-II)

Constitutes 15-20% of HAE patients. The level of C1 Inhibitor may be either normal or elevated; however, the function
of the C1 Inhibitor is compromised. Again, the levels of C3 and C1q are normal, while C4 is low. HAE-II also does not
respond to antihistamines or corticosteroids.

Hereditary Angioedema with Normal C1 Inhibitor

Number of cases currently unknown. Patients must have a family history of angioedema. C1 Inhibitor levels and func-
tion are both normal. Although a minority of cases have a mutation in the coagulation factor XII gene, this mutation
has not been shown to cause the disease. The disease predominantly affects females; with the swelling sometimes
associated with pregnancy or the use of estrogen-containing contraceptives. As with HAE-I and HAE-II, this form of
the disease does not respond to antihistamines or corticosteroids.4

Acquired Angioedema
There are two forms of acquired angioedema. The first, AAE-I, can be seen in patients with lymphoma or other car-
cinomas and is characterized by significantly diminished levels of C1q, in addition to low C1 Inhibitor.6 The second,
AAE-II, is caused by auto-antibodies to C1 Inhibitor that interfere with its function. Treatment for these conditions is
elimination of the underlying causes of the angioedema through the use of chemotherapy (in the case of cancer) or
immunosuppression (in the case of auto-antibodies) [3].

Anabolic steroids is the most commonly prescribed preventative treatment for HAE. Unfortunately, steroid treatment
cannot be used in children and is not well tolerated by many women. It can also lead to liver toxicity and an increase
in cholesterol levels. There are currently two FDA-approved C1 Inhibitor products available for treatment of HAE. Cin-
ryze™ (ViroPharma) is a C1 acute abdominal, facial and laryngeal attacks.

REFERENCES

[1] Landerman NS. Hereditary angioneurotic edema, I: case re- [4] Glovsky MM et al. Complement determinations in human
ports and review of the literature. J. Allergy. 33:316-341 (1962). disease. Ann. Allergy Asthma Immunol. 93:513-523 (2004).

[2] Donaldson VH and Evans RR. A biochemical abnormality in
hereditary angioneurotic edema: Absence of serum inhibitor of C’
1-esterase. Am. J. Med. 35:37-44 (1963).
[3] Lachmann PJ. Complement deficiencies: genetic and ac-
quired. In: Clinical Aspects of Immunology, 5th ed. (1993).
[5] US Hereditary Angioedema Association. In: What is Hereditary
Angioedema? http://www.haea.org/patients/what-is-hae/ (2012)
[6] Caldwell JR et al. Acquired C1 inhibitor deficiency in lympho-
sarcoma. Clin. Immunol. Immunopathol. 1:39-59 (1972).

11 TECOmedical
ISCHEMIA/REPERFUSION(I/R)INJURY
Ischemia is a restriction in blood supply to a tissue resulting in a lack of oxygen and glucose required for cellular me-
tabolism. Depending on the length and severity of the ischemia, toxic compounds can accumulate intracellularly and
cause loss of organ function. In addition to ischemia injury caused by different types of vascular occlusion (such as
stroke, myocardial infarction or Sickle cell disease), there is also a major impact during organ transplantation. When
circulation is re-established, either by removal of the occlusion or transplant of an organ, oxygen is reintroduced to
the tissues and repair mechanisms are triggered. Flushing of the accumulated toxins into the system can affect other
organs or negatively influence the regeneration of the ischemic organ. Major components of ischemia reperfusion
injury (IR) include neutrophil stimulation and complement activation [1,2]. Complement activation during myocardial
infarction was described in the 1970’s [3].

Complete depletion of complement components and inhibition of specific pathway components have both been
used in the attempt to identify drug targets for IR. Most of these attempts have been directed at the classical and
lectin complement pathways. Early studies of IR following myocardial infarction used Cobra Venom Factor (CVF) to
completely deplete the C3 complement protein [4]. However, due to immunogenicity issues, this treatment has not
been used clinically. In 2007, a humanized form of CVF, HC3-1496, was developed which depleted C3. This compound
was effective in reducing the infarct size and preserving cardiac function in a mouse model [5]. Inhibition of comple-
ment activation has also been shown to reduce IR of various organs [6,7,8,9].

One of the first specific inhibitors developed for clinical use was recombinant, soluble Complement Receptor 1 (sCR1),
which showed beneficial effects in models of myocardial infarction, experimental lung and liver transplantation and
intestinal IR. It was suggested that sCR1 may also provide protection during stroke and injury of skeletal muscle. Ho-
wever, it has not advanced into further clinical trials.

C1 Inhibitor has been shown to have protective effects in both myocardial infarction and lung transplantation. A no-
vel C1s inhibitor and a chimeric inhibitor derived from human Decay Acceleration Factor ( DAF, CD55) and Membrane
Co-factor Protein (MCP, CD46) have also been used to block classical/lectin pathway activation models of myocardial
infarction (C1s Inhibitor) [10] and xenograft transplantation (chimeric inhibitor) [11]. Attempts to block the lectin pa-
thway using monoclonal antibodies against rat mannose binding lectin have also shown reduction in post-ischemic
myocardial infarction injury in rats [12].

Inhibitors and blocking antibodies of C5 and C5a are another target of therapeutic research. Blockade of C5 and C5a
provided protection in a rat intestinal IR model [13]. Antibodies to C5 significantly reduced necrosis and cell apoptosis
following myocardial infarction in rats [14]. In pigs, the use of antibodies against C5a reduced both myocardial injury
and cardiac endothelial dysfunction after IR [15].

It is possible that complement activation pathways in IR may be organ dependent, since C4 knockout mice have no
significant protection from kidney IR (C3, C5 and C6 knockout mice were protected) [16]; while in earlier studies, both
C3 and C4 knockout mice were protected from injury in skeletal muscle and intestine [17].

Recently, the role of complement activation in the pathogenesis of whole organ ischemia highlighted the predo-
minant role of lectin molecules in several organ models of I/R injury [18]. One possible trigger mechanism leading
to activation of the lectin pathway is the binding of natural IgM to epitopes exposed on ischemic tissue, with no
involvement of previously implicated classical pathway activation. The alternative pathway is implicated in some ins-
tances, for example following renal ischemia, and may serve to amplify the cleavage of C3 and subsequent evolution
of injury, following initiation through the lectin pathway, or could be seen as an independent event. This prevalent
role of lectin pathway activation makes it an attractive target for therapeutic intervention, with MASP-2a potential
candidate because of an absolute requirement of MASP-2 for lectin pathway activation in the pathogenesis of renal
reperfusion injury. These findings justify the investigation of therapeutic blockade of lectin pathway activation during
the immediate post-transplant stage, where ischemic events have a significant impact upon renal allograft function.

12
 REFERENCES

[1] Riedmann NC and Ward PA. Complement in ischemia reper-
fusion injury. Am. J. Path. 162(2):363-367 (2003).
[2] Chan RK et al. Ischaemia-reperfusion is an event triggered
by immune complexes and complement. Brit J. Surg. 90:1470-1478
(2003).
[3] Hill JH and Ward PA. The phlogistic role of C3 leukotactic
fragments in myocardial infarcts of rats. J. Exp. Med. 133:885-900
(1971).
[4] He S et al. A complement-dependent balance between
hepatic ischemia/reperfusion injury and liver regeneration in
mice. J. Clin. Invest. 119(8):2304-2316 (2009).
[5] Gorsuch WB et al. Humanized cobra venom factor decreases
myocardial ischemia-reperfusion injury. Mol. Immunol. 47:506-510
(2009).
[6] Hofmann U et al. Nothing but natural: Targeting natural IgM
in ischaemia/reperfusion injury. Cardio. Res. 87:589-590 (2010).
[7] Tofferi J et al. C3b-independent complement activation in
ischemia/reperfusion mesenteric tissue injury in autoimmune
prone (B6.MRL/lpr) mice. ISRN Immunol. 2012.
[11] Kroshus TJ et al. A recombinant soluble chimeric comple-
ment inhibitor composed of human CD46 and CD55 reduces acute
cardiac tissue injury in models of pig to human heart transplanta-
tion. Transplantation 69:2282-2289 (2000).
[12] Jordan JE et al. Inhibition of manose-binding lectin reduces
postischemic myocardial reperfusion injury. Circulation 104:1413-
1418 (2001).
[13] Arumgam TV et al. Protective effect of new C5a receptor
antagonist against ischemia-reperfusion injury in the rat small
intestine. J. Surg. Res. 103:260-267 (2002).
[14] Vakeva AP et al. Myocardial infarction and apoptosis after
myocardial ischemia and reperfusion: role of the terminal com-
plement components and inhibition by anti-C5 therapy. Circula-
tion 97:2259-2267 (1998).
[15] Tofukuji M et al. Anti-C5a monoclonal antibody reduces
cardiopulmonary bypass and cardiopledia-induced coronary en-
dothelial dysfunction. J. Thorac. Cardiovasc. Surg. 116:1060-1068
(1998).
[16] Zhou W et al. Predominant role for C5b-9 in renal ischemia/
reperfusion injury. J. Clin. Invest. 105:1363-1371 (2000).

[8] Arumugam TV et al. Complement mediators in ischemia- [17] Williams JP et al. Intestinal reperfusion injury is mediated by
reperfusion injury. Clin. Chim. Acta. 274:33-45 (2006). IgM and complement. J. Appl. Physiol. 86:938-942 (1999).

[9] Gorsuch WB et al. The complement system in ischemia- [18] Conrad A et al. Which pathways trigger the role of comple-
reperfusion injuries. Immunobio. 217:1026-1033 (2012). ment in ischemia/reperfusion injury? REVIEW ARTICLE.
Frontiers in immunology. Volume3: Article 341 (2012).
[10] Buerke M et al. Novel small molecule inhibitor of C1s exerts
cardioprotective effects in ischemia-reperfusion injury in rabbits.
J. Immunol. 167:5375-5380 (2001).

RHEUMATOID ARTHRITIS
Rheumatoid Arthritis (RA) is a chronic, progressive and disabling autoimmune disease affecting 4.4 million people
worldwide. It affects approximately 3 times more women than men. Disease onset can occur at any age but is gene-
rally between 30-50 years of age. The disease is associated with high morbidity and daily activities are significantly
impaired in most individuals. After 5 years of disease, approximately 33% of sufferers will not be working and after 10
years, approximately half will have substantial functional disability [1]. There is no known cure for RA, but many dif-
ferent types of treatment can alleviate symptoms and/or modify the disease process. Several major pharmaceutical
companies have therapeutics available: Embrel (Amgen/Pfizer), Remicade and Simponi (Centocor Ortho Biotech), Hu-
mira (Abbott), Cimzia (USB Group), Rituximab (Roche), Orencia (Bristol Myers Squibb), etc. A combination of imaging
studies, blood tests and physical examinations are used to diagnose Rheumatoid Arthritis.

In 1965, Schubart et al. showed that serum complement levels may fluctuate significantly during active rheumatoid
arthritis [2]. Versey et al. demonstrated that complement conversion (the breakdown of C3 and C4) was more frequent
in patients with rheumatoid arthritis than in normal individuals [3]. Studies have shown high levels of complement
components C3, C3a, C5a, and TCC (C5b-9) in serum, synovial fluid and synovial tissue of patients with RA [4-6]. Eleva-
ted levels of C1q-C4 complexes are also correlated with disease activity [7]. It is theorized that complement activation
is induced (at least in part) by immune complexes consisting of type II collagen (CII) and CII autoantibodies [8]. Spe-
cific modulation or inhibition of local complement production (in the synovial tissue) could be a viable target for RA
therapy [9].

Studies are now looking at the correlation between components of complement activation and other proteins com-
monly associated with RA. CRP can form complexes with C3d and C4d and increased levels of these complexes are

13 TECOmedical
seen in RA patients. Treatment with infliximab (a TNFαblocker) significantly reduces the level of CRP and the CRP
complexes [10]. ACPA activate both the classical and alternative complement pathways. Data suggests that at high
serum levels of ACPA, the alternative pathway may be more involved than the classical [11]. Cartilage Oligomeric
Matrix Protein (COMP), a protein which is elevated in the serum of patients with active joint disease (such as RA), may
activate the alternative pathway through an interaction with properdin. COMP was also shown to inhibit the classical
and lectin pathways. Detection of COMP-C3b complexes may provide an early marker of RA, which would allow for
early treatment and avoidance of extensive cartilage damage [12].

REFERENCES

[1] Frequencies of selected diseases and conditions among
persons 18 years of age and over, by selected characteristics (Ta-
ble 7). In: National Health Interview Survey. Centers for Disease Control
and Prevention (2008).
[2] Schubart AF et al. Serum complement levels in rheumatoid
arthritis. Ann Rheum. Dis. 24:439-450 (1965).
[3] Versey JMB et al. Complement metabolism in rheumatoid
arthritis. Ann Rheum Dis. 32:557-564 (1973).
[4] Ruddy S et al. Rheumatoid arthritis biosynthesis of comple-
ment proteins by synovial tissues. N. Engl. J. Med. 290:1284-1288
(1974).
[5] Jose, PJ et al. Measurement of the chemotactic complement
fragment C5a in rheumatoid synovial fluids by radioimmunoas-
say: Role of C5a in the acute inflammatory phase. Ann. Rheum. Dis.
49:747-752 (1990).
[6] Morgan BP. The complement system: An overview. Methods
Mol. Biol. 150:1-13 (2000).
[7] Wouters, D et al. Evaluation of classical complement pathway
activation in rheumatoid arthritis. Arthritis Rheum. 54(4):1143-
1150 (2006).
[8] Watson WC et al. Assessment of the potential pathogenicity
of type II collagen autoantibodies in patients with rheumatoid
arthritis: Evidence of restricted IgG3 subclass expression and ac-
tivation of complement C5 to C5a. Arthritis Rheum. 29:1316-1321
(1986).
[9] Neumann E et al. Local production of complement protins
in rheumatoid arthritis synovium. Arthritis Rheum. 46(4):934-945
(2002).
[10] Familian, A et al. Infliximab treatment reduces complement
activation in patients with rheumatoid arthritis. Ann. Rheum. Dis.
64:1003-1008 (2005).
[11] Trouw LA et al. Anti-cyclic citrullinated peptide antibodies
from rheumatoid arthritis patients activate complement via both
the classical and alternative pathways. Arthritis Rheum. 60(7):1923-
1931 (2009).
[12] Happonen, KE et al. Regulation of complement by cartilage
oligomeric matrix protein allows for a novel molecular diagnostic
principle in rheumatoid arthritis. Arthritis Rheum. 62(12):3574-3583
(2010).

HEMOLYTIC UREMIC SYNDROME

Hemolytic Uremic Syndrome (HUS) is characterized by hemolytic anemia (caused by the destruction of red blood
cells), acute kidney failure (uremia) and a low platelet count (thrombocytopenia). The syndrome, which predominant-
ly effects children, was first described in 1955 [1]. There are two forms of the disease, Shiga toxin-producing E. coli HUS
(STEC-HUS) and atypical hemolytic uremic syndrome (aHUS). The more common, STEC-HUS, is triggered by bacterial
infection [2]. aHUS (a more rare and severe form) is the result of genetic defects which cause chronic, uncontrolled
complement activation [3]. Both manifestations of the disease cause endothelial damage, leukocyte activation, pla-
telet activation, and widespread inflammation and multiple thrombosis in the small blood vessels (a condition called
systemic thrombotic microangiopathy; TMA) which can lead to organ damage/failure and death [4].

Shiga toxin-producing E. coli Hemolytic Uremic Syndrome (STEC-HUS)

Accounts for approximately 90% of HUS cases. [5]. STEC-HUS is associated with infection by bacteria producing shiga
toxins (Stx1 and Stx2), most often O157:H7 serotype enterohaemorrhagic E. coli [6]. Increased levels of the break-
down products of two components of the alternative pathway, C3 (C3b, C3c, C3d) and Factor B (Ba), were found in
the plasma of children who were likely to have had STEC-HUS [7]. In 2009, a study of 17 children in the acute phase
of STEC-HUS showed elevated plasma levels of Bb and SC5b-9 [8]. Mice deficient in Factor B that were treated with
Stx2 and lipopolysaccharide (LPS) exhibited less thrombocytopenia and were protected against impairment of renal
function [9]. These findings indicate activation of the alternative pathway in the pathobiology of STEC-HUS.

14
 Atypical Hemolytic Uremic Syndrome (aHUS)
Aaccounts for <10% of HUS cases. aHUS is the result of defects in the genes coding for complement regulatory pro-
teins [10]. Warwicker et al. (1998) reported an association between a mutation in Factor H (CFH) and aHUS [11]. Since
that time, more than 120 mutations have been identified in Factor H and other genes, such as those coding for mem-
brane cofactor protein (MCP), Factor I (CFI), and thrombomodulin (THBD) (Figure 7) Autoantibodies against Factor
H have also been described in aHUS patients, mainly in children lacking Complement Factor H –related protein 1
(CFHR1) [12]. In addition, mutations in two components of the alternative complement pathway, C3 and Factor B
(CFB), have been identified [13]. Patients with Factor H mutations have the worst prognosis, with 70-80% developing
end-stage renal disease (ESRD) or death. They also have a high percentage of post-transplantation recurrences. Al-
ternatively, those with MCP mutations rarely develop ESRD and, if they do require transplant, usually have excellent
outcomes [14].

The most common treatment for aHUS is plasma exchange or plasma infusion. However, some patients are resistant
to these treatments. Eculizimab (Solaris®, Alexion Pharmaceuticals), a humanized monoclonal antibody against C5,
has recently shown to be an effective treatment for aHUS. Eculizimab, currently used as a treatment for paroxysmal
nocturnal hemoglobinuria (PNH), is now in clinical trials to determine its efficacy and safety for maintenance of re-
mission of hemolysis and thrombocytopenia in both plasma therapy sensitive and plasma therapy resistant aHUS
patients [14].

Figure 7. Summary of clinical outcome in patients with aHUS [14].

REFERENCES

[1] Gasser C et al. Hemolytic-uremic syndrome: Bilateral necro- [8] Thurman JM et al. Alternative pathway of complement in
sis of the renal cortex in acute acquired hemolytic anemia. Schweiz children with diarrhea-associated hemolytic uremic syndrome.
Med. Wochenschr. 85:38-39 (1955). Clin. J. Am. Soc. Nephrol. 4:1920-1924 (2009).

[2] Shimizu Y et al. Thomsen-Friedenreich antigen exposure as [9] Morig, M et al. Alternative pathway activation of comple-
a cause of Streptococcus pyogenes-associated hemolytic-uremic ment by Shiga toxin promotes exuberant C3a formation that trig-
syndrome. Clin. Nephrol. 78(4):328-331 (2012). gers microvascular thrombosis. J. Immunol. 187:172-180 (2011).

[3] Loirat C et al. Complement and the atypical hemolytic ure- [10] Noris M et al. STEC-HUS, atypical HUS and TTP are all disea-
mic syndrome in children. Pedatr. Nephrol. 23(11):1957-72 (2008). ses of complement activation. Nat. Rev. Nephrol. 8:622-633 (2012).

[4] Zipfel PF et al. Thrombotic microangiopathy: New insights [11] Warwicker P et al. Genetic studies into inherited and spora-
and challenges. Curr. Opin. Nephrol. Hypertens. 19:242-247 (2010). dic hemolytic uremic syndrome. Kidney Int. 53:836-844 (1998).

[5] Hirt-Minkowski, P et al. Atypical hemolytic uremic syndrome:
Update on the complement system and what is new. Nephron. Clin.
Pract. 114:c219-c235 (2010).
[6] Tarr PI et al. Shiga-toxin-producing Escherichia coli and he-
molytic uraemic syndrome. Lancet 365:1073-1086 (2005).
[12] Noris, M and Renuzzi, G. Atypical hemolytic uremic syndro-
me. N. Engl. J. Med. 361:1676-1687 (2009).
[13] Lee BH et al. Atypical hemolytic uremic syndrome associa-
ted with complement Factor H autoantibodies and CFHR1/CFHR3
deficiency. Pediatr. Res. 66:336-340 (2009).

[7] Monnens L et al. The complement system in hemolytic-ure- [14] Waters AM and Licht C. aHUS caused by complement dysre-
mic syndrome in childhood. Clin. Nephrol. 13:168-171 (1980). gulation: New therapies on the horizon. Pediatr. Nephrol. 26:41-57
(2011).
15 TECOmedical
PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease characterized by complement-induced in-
travascular hemolytic anemia (due to destruction of red blood cells), red urine (due to the presence of hemoglobin in
the urine) and thrombosis. PNH is rare and potentially life-threatening. The complement-mediated lysis of red blood
cells, neutrophils and platelets is most often seen at night because respiratory acidosis enhances attachment of com-
plement components to the cells [1].

PNH is caused by an acquired (rather than inherited) intrinsic defect of myoid stem cells. One or several hemotopoietic
stem cells have a somatic mutation of the X-chromosome gene, PIGA, required for synthesis of the glycosyl phophati-
dylinositol (GPI) moiety that anchors some proteins to the cell surface. The progeny of effected cells are deficient in all
the GPI-anchor proteins (GPI-AP) that are normally expressed on the cells. Among these proteins are DAF (CD55) and
MIRL (CD59), the two primary erythrocyte membrane regulators of complement [2]. PNH III cells are completely de-
ficient in GPI-AP’s, PNH II cells are partially (~90%) deficient and PNH I cells express GPI-AP’s at normal density. Based
on flow cytometry, patients with a high percentage of type III cells have high grade hemolysis. Patients with a high
percentage of type II cells, but a low percentage of type III cells and patients with a low percentage of type III cells have
minimal hemolysis [3]. Additional tests required for evalulation of a patient with PNH include: complete blood count
(to assess effect on production of leukocytes, platelets and erythrocytes), serum concentration of lactate dehydro-
genase (LDH), bilirubin and haptoglobin (biochemical markers of hemolysis), determination of iron stores, bone mar-
row aspirate and biopsy, and cytogenetics. These studies will allow classification of the patient into a PNH category [4].
The International PNH Interest Group has identified three categories of PNH: 1. Classic PNH, 2. PNH in the setting of
another bone marrow (BM) failure syndrome, and 3. Subclinical PNH [5].

Subclinical PNH
Approximately 60% of patients with aplastic anemia and 20% of patients with low risk myelodysplastic syndrome
(MDS) have detectable levels of GPI-AP deficient erythrocytes and granulocytes. Among these patients, ~80% have
a proportion of GPI-AP deficient cells <1.0% of the total. These patients are classified as subclinical PNH; having no
clinical or biochemical evidence of hemolysis and requiring no special treatment for PNH.
PNH in the setting of another BM failure syndrome – Patients with a BM failure syndrome (such as aplastic anemia
or MDS) and clinical or biochemical evidence of hemolysis are classified as PNH in the setting of another BM failure
syndrome. Most patients with PNH/AA or PNH/MDS have a small number of PNH cells (<10%) and require no special
treatment. Treatment is usually focused on the underlying BM failure syndrome and not PNH.

Classic PNH
Patients have a large number of PNH cells (>50%) and have extensive intravascular hemolysis (indicated by elevated
serum LDH). Symptoms include episodic hemoglobinuria, lethargy, malaise and asthenia that can be debilitating.

Treatment
The only cure for PNH is allogenic bone marrow transplant. Until 2007, transplantation was the primary treatment for
patients with bone failure, life-threatening thrombosis and uncontrollable hemolyis. Studies determined that com-
plement-mediated hemolysis can be inhibited by blocking formation of the membrane attack complex (MAC). Eculi-
zumab (Solaris®, Alexion Pharmaceuticals) is a humanized monoclonal antibody that binds the complement protein,
C5. Eculizumab prevents the cleavage of C5 to C5b by the C5 convertase of the alternative complement pathway;
inhibiting formation of the MAC. Patients experience a reduction in the incidence of thrombosis, improved renal
function and reduction in nitric oxide comsumption (which can cause symptoms such as recurrent abdominal pain,
dysphagia, erectile dysfunction and severe lethargy) [6-7]. Because Eculizumab inhibits the terminal complement pa-
thway, patients can be at increased risk of developing infections with encapsulated organisms, particularly Neisseria
meningitides or Neisseria gonorrhoeae [8]. The incidence of meningococcal infection appears to be small (~0.5 cases
per 100 patient years on Eculizumab) [9-10].

16
 REFERENCES

[1] Kumar V et al. Hemolytic Anemia. In: Robbins Basic Pathology. [7] Hillmen P. The role of complement inhibition in PNH. Am.
8th ed. pg. 432 (2007). Soc. Hematol. Educ. Program 2008:116-123 (2008).

[2] Parker CJ. Hemolysis in PNH. In: Paroxysmal nocturnal he-
moglobinuria and the glycosylphosphatidylinositol-linked pro-
teins. Young, NS and Moss, J (eds.) 49-100 (2000).
[3] Parker CJ. The pathphysiology of paroxysmal nocturnal he-
moglobinuria. Exp. Hematol. 35:523-533 (2007).
[4] Parker CJ. Management of paroxysmal nocturnal hemoglo-
binuria in the era of complement inhibitory therapy. Am. Soc. He-
matol. Educ. Program 2011:21-9 (2011).
[5] Parker CJ et al. Diagnosis and management of paroxysmal
nocturnal hemoglobinuria. Blood 106:3699-3709 (2005).
[8] Ross SC and Densen P. Complement deficiency states and
infection: Epidemiology, pathogenesis and consequences of neis-
serial and other infections in an immune deficiency. Medicine (Bal-
timore) 63:243-273 (1984).
[9] Schubert J et al. Eculizumab, a terminal complement inhi-
bitor, improves anaemia in patients with paroxysmal nocturnal
haemoglobinuria. Br. J. Haematol. 142(2):263-72 (2008).
[10] Hillmen P et al. Effect of the complement inhibitor eculizu-
mab on thromboembolism in patients with paroxysmal nocturnal
hemoglobinuria. Blood 110(12):4123-4128 (2007).

[6] Parker CJ. Eculizumab for paroxysmal nocturnal haemoglo-

binuria. Lancet 373:759-767 (2009).

MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS

Glomerulonephritis (GN) is a renal disease characterized by inflammation of the glomeruli (small blood vessels in the
kidney) [1]. Membranoproliferative glomerulonephritis (MPGN) is caused by the buildup of immune complexes or
other material in the kidney glomerular mesangium and basement membrane (GBM) [2]. The GBM helps filter waste
and extra fluid from the blood. Damage to the membrane affects the kidney’s ability to create urine. It may allow
blood and protein to leak into the urine, which can eventually lead to edema. Most cases of MPGN are caused by
autoimmune diseases (SLE, scleroderma, etc.), cancer (leukemia, lymphoma), or infections (hepatitis B, malaria, etc.).
MPGN mainly affects children (ages 8 to 16).

THREE TYPES OF MPGN

Type I
Most common form of MPGN is caused by the deposition of immune complexes in the kidney [3]. It is characterized
by subendothelial and mesangial immune deposits. Type I MPGN is believed to be mediated by the classical pathway
of complement (low or normal C3, low C4 and CH50) [4]. The disruption of the C1q in mice leads to spontaneous de-
velopment of glomerulonephritis [5].

Type II
Also called “dense deposit disease”, Type II accounts for <20% of MPGN in children and is even less common in adults.
Within 10 years, at least one-half of people diagnosed with Type II MPGN have progressed to end-stage renal disease.
Unlike Type I MPGN, the deposits are not immune complexes. Instead, they are more similar to the drusen seen
between the Bruch’s membrane and the retinal pigment epithelium (RPE) of patients with AMD [6]. Also, unlike Type
I, Type II MPGN is mediated by the alternative complement pathway (low C3, normal C4 and low CH50). Greater than
80% of patients with MPGN II are positive for serum C3 nephritic factor (C3NeF), an autoantibody directed against
C3bBb, the C3 convertase of the alternative pathway [7].

Type III
Most rare form of the disease. Complement activation of both the alternative and terminal complement pathways
(low C3, normal C4 and low C5 – C9) in type III MPGN is thought to be related to a slow-acting nephritic factor (termed
the nephritic factor of the terminal pathway (NeFt) ) that stabilizes a properdin dependent C5-convertase, (Cb3)2BbP,
thus activating the terminal pathway [8]. This nephritic factor has not been reported in healthy subjects, unlike C3NeF.
In addition, the deposits observed in renal biopsies of patients with type III MPGN are closely associated with hypo-
complementemia, suggesting that NeFt is fundamental to the pathogenesis of type III MPGN [9].

17 TECOmedical
Treatments for MPGN have included corticosteroids, immunosuppressives, antiplatelet regimens, plasma exchange
and biologic agents. Future treatments which directly impact the complement system, such as Rituximab (a mono-
clonal antibody that targets CD20 on the surface of B lymphocytes) to reduce C3NeF or Eculizumab (a humanized
anti-C5 monoclonal antibody currently used to treat PNH), along with new therapies that will reduce the deposition
seen with the disease [10].

REFERENCES

[1] Hricik DE et al. Glomerulonephritis. N. Engl. J. Med. 339(13):888- rulonephritis type II (“dense deposit disease”). Am. J. Kidney Dis.
899 (1998). 42(2):E2–5 (2003).

[2] Membranoproliferative GN. PubMed Health. www.ncbi.nlm.
nih.gov/pubmedhealth/PMH0001507/ (2012).
[3] Berger SP et al. Complement and the kidney: What the
nephrologist needs to know in 2006? Nephrol. Dial. Transplant.
20:2613-2619 (2005).
[4] Varade WS et al. Patterns of complement activation in idio-
pathic membranoproliferative glomerulonephritis, types I, II, and
III. Am. J. Kidney Dis. 16:196–206 (1990).
[5] Botto M et al. Homozygous C1q deficiency causes glomeru-
lonephritis associated with multiple apoptotic bodies. Nat. Genet.
19:56-59 (1998).
[6] Colville D et al. Visual impairment caused by retinal abnor-
malities in mesangiocapillary (membranoproliferative) glome-
[7] Schwertz R et al. Complement analysis in children with idio-
pathic membranoproliferative glomerulonephritis: A longterm
follow-up. Pediatr. Allergy Immunol. 12: 166–172 (2001).
[8] Braun MC and Strife CF. Membranoproliferative glomerulo-
nephritis. In: Pediatric nephrology and urology: the requisites in
pediatrics. Kaplan BS, Meyers KEC, (eds.) pp. 147–155 (2004).
[9] West CD and McAdams AJ. Membranoproliferative glome-
rulonephritis type III: Association of glomerular deposits with
circulating nephritic factor-stabilized convertase. Am. J. Kidney
Dis. 32:56–63 (1998).
[10] Alchi B and Jayne D. Membranoproliferative glomerulo-
nephritis. Pediatr. Nephrol. 25(8): 1409–1418 (2010).

ALZHEIMER’S DISEASE
Alzheimer’s disease (AD) is a form of dementia that has no cure, worsens as it progresses, and eventually leads to
death. It was first described by German psychiatrist and neuropathologist, Alois Alzheimer, in 1906 [1]. Most often,
AD is diagnosed in people over 65 years of age, although the less-prevalent early-onset Alzheimer’s can occur much
earlier. In 2006, there were 26.6 million sufferers worldwide. Alzheimer’s is predicted to affect 1 in 85 people globally
by 2050 [2].

The cause and progression of Alzheimer’s disease are not well understood. Recent studies involved in identifying
biomarkers for AD correlated elevated cerebrospinal fluid (CSF) of complement proteins, C3 and C4, in patients with
dementia compared with patients suffering from mild cognitive impairment which did not progress to AD [3]. Com-
plement activation in AD is thought to be triggered by the interaction of complement proteins with aggregated
forms of amyloid-beta (Aβ) and tau protein, the major components in plaques and neurofibrillary tangles (NFTs). Ag-
gregated Aβ binds C1q, activating the classical complement pathway [4]. See table below for more information on the
biological activity of different complement components. In addition, Factor B mRNA has been observed in the frontal
cortex of the AD brain [5], indicating that the alternative pathway may also be activated.

Inhibitors of the complement system are reduced in AD. C1 Inhibitor is reduced in the plasma of AD patients, which
may be the result of the inability of neurons and astrocytes to secrete the active form of the protein [6,7].
Microglia, the immune cells of the brain, become reactive and are implicated in neuronal loss and cognitive decline
in AD. Evidence suggests that microglia in AD are influenced by complement factors to adopt either protective or
harmful phenotypes [8].

Since there is increasing evidence of the involvement of the complement system in AD, an accurate biomarker uti-
lizing this system that has significant diagnostic or predictive value would be an excellent tool for developing and
testing novel therapies for the treatment of Alzheimer’s Disease [10].

18
 Figure 8. Biological activities of complement proteins related to Alzheimer’s Disease [9].

REFERENCES

[1] Berchtold NC and Cotman CW. Evolution in the conceptuali-
zation of dementia and Alzheimer’s disease: Greco-Roman period
to the 1960s. Neurobiol. Aging. 19(3):173-178 (1998).
[2] Brookmeyer R et al. Forecasting the global burden of Alzhei-
mer’s disease. Alzheimer’s and Dementia 3(3):186–91 (2007).
[3] Daborg J et al. Cerbrospinal fluid levels of complement
proteins C3, C4 and CR1 in Alzheimer’s disease. J. Neural Trans.
119(7):789-797 (2012).
[4] Loeffler DA. Significance of complement activation in Alz-
heimer’s disease. Touch Briefings pgs. 52-55 (2008).
[5] Strohmeyer, R et al. Detection of complement alternative pa-
thway mRNA and proteins in the Alzheimer’s disease brain. Mol.
Brain Res. 81:7-18 (2000).
[6] Zhang R et al. Mining biomarkers in human sera using prote-
omic tools. Proteomics 4(1):244-256 (2004).
[7] Veerhuis, R et al. Complement C1-inhibitor expression in Alz-
heimer’s disease. Acta Neuropath. 96(3):287-296 (1998).
[8] Crehan H et al. Microglia, Alzheimer’s disease, and comple-
ment. Int. J. Alzheimer’s Dis. 2012:1-10 (2012).
[9] Loeffler DA. Using animal models to determine the signifi-
cance of complement activation in Alzheimer’s disease. J. Neuroin-
flamm. 1:18-30 (2004).
[10] Aiyaz M et al. Complement activation as a biomarker for Alz-
heimer’s disease. Immunobio. 217:204-215 (2012).

CANCER
Chronic inflammatory processes always include futile repair processes including cell protective mechanisms that
eventually will support a cancerogenic transformation. It is widely believed that the chronicity of inflammation de-
termines its cancer effect: Acute inflammation is believed to fight the development of neoplastic cells, whereas chro-
nic inflammation encourages their genesis and spread (1-3]. The complement cascade contains some of the most
powerful proinflammatory molecules in the body, including anaphylatoxins C3a and C5a [1-5]. The contribution of
the complement cascade to acute inflammation is well established, as is the continuous activation and consumption
of complement proteins in chronic inflammatory states [4, 5]. Findings that complement proteins C3, C4, and C5a
may aid tumor growth through immunosuppression [6] suggests an insidious and previously unrecognized relation-
ship between the complement system and cancer[7]. Rutkowski et al. [7] presented ample evidence of the diverse
means by which complement proteins may facilitate the major features of carcinogenesis, including dysregulation of
mitogenic signaling pathways, sustained cellular proliferation, angiogenesis, insensitivity to apoptosis, invasion and
metastasis, and escape from immunosurveillance.

At present, a number of studies are exploring the potential role for complement-mediated therapeutics in the fight
against cancer. There is particular interest in the use of monoclonal antibodies against the soluble and membrane-
bound complement inhibitors displayed by various tumors [1], a strategy believed to enhance antitumor comple-
ment-dependent cytotoxicity and antibody dependent cell-mediated cytotoxicity. Despite some promising early re-
sults [8-10], such strategies ignore the possibility that the complement system promotes neoplastic development and
progression rather than exclusively retarding it. Ultimately, the increasingly complex picture of complement interac-
tion with cancer demands further study, but does offer the intriguing possibility that anti-complement strategies may
offer an entirely new means of fighting cancer.

19 TECOmedical
REFERENCES

[1] Markiewski MM et al. Is complement good or bad for cancer
patients? A new perspective on an old dilemma. Trends Immunol
30:286–92 (2009).
[2] Loveland BE et al. J. Cancer exploiting complement: a clue or
an exception? Nat Immunol 9:1205–6 (2008)
[3] Ostrand-Rosenberg S. Immune surveillance: a balance
between protumor and antitumor immunity. Curr Opin Genet Dev
18 (2008).
[4] Guo RF, Ward PA. Role of C5a in inflammatory responses.
Annu Rev Immunol 23:821–52 (2005).
[5] Kohl J. Anaphylatoxins and infectious and non-infectious
inflammatory diseases. Mol Immunol 38:175–87 (2001).
[7] Rutkowski MJ, Sughrue ME, Kane AJ, et al. Cancer and the
Complement Cascade. Mol Cancer Res 8:1453-1465 (2010).
8] Gelderman KA et al. Enhancement of the complement activa-
ting capacity of 17-1A mAb to overcome the effect of membrane-
bound complement regulatory proteins on colorectal carcinoma.
Eur J Immunol. 32: 128–35 (2002).
[9] Sier CF et al. β-Glucan enhanced killing of renal cell carci-
noma micrometastases by monoclonal antibody G250 directed
complement activation. Int J Cancer 109:900–8 2004).
[10] Allendorf DJ et al. C5a-mediated leukotriene B4-amplified
neutrophil chemotaxis is essential in tumor immunotherapy fa-
cilitated by anti-tumor monoclonal antibody and β-glucan. J Im-
munol 174:7050–6 (2005).

[6] Markiewski MM et al. Modulation of the antitumor immune

response by complement. Nat Immunol 9: 1225–35 (2008).

3 OTHER DISEASES
3.1 LYME DISEASE
Lyme disease is caused by the spirochete Borrelia burdgorferi and is spread by the bite of ticks. It is the most common
tick-borne illness in the world today. Recommendations from the Centers for Disease Control and Prevention for la-
boratory testing include an enzyme-linked Immunosorbent assay (ELISA) followed by confirmation of positive results
by Western blot [1]. However, these tests are problematic and can result in false negatives if testing is performed too
soon after receiving the bite. Shoemaker et al reported increased levels of the complement protein fragments, C3a
and C4a, in patients with acute Lyme disease [2]. Additional studies have shown that patients with predominant mus-
culoskeletal symptoms of Lyme disease have normal C3a and elevated C4a levels. Changes in C4a correlate with the
response to therapy in chronic Lyme disease (response to treatment lowers C4a levels) [3]. Therefore, C4a may be a
valuable marker in patients with persistent symptoms of Lyme disease.

3.2 ALLERGIC ASTHMA

Allergic asthma is a chronic inflammatory disease of the upper airway. It arises as a result of inappropriate immune
responses to common environmental antigens (pollutants, cigarette smoke, allergens, etc.) in certain individuals [4].
There is evidence that complement is activated in the lungs of individuals with asthma. The anaphylatoxins, C3a and
C5a were elevated after allergen challenge to individuals with asthma, while they remained unchanged in normal
individuals [5,6]. The balance between C3a and C5a production may determine the tendency to develop immunity/
tolerance to inhaled allergens. Further study is needed to completely elucidate the relationship between the anap-
hylatoxins and asthma [7].

REFERENCES
[5] Krug N et al. Complement factors C3a and C5a are increased
[1] Aguero-Rosenfeld ME et al. Diagnosis of lyme borreliosis. Clin.
in bronchoalveolar lavage fluid after segmental allergens provocation
Microbiol. Rev. 18:484-509 (2005).
in subjects with asthma. Am. J. Respir. Crit. Care Med. 164:1841-1843
(2001).
[2] Shoemaker RC et al. Complement split products C3a and C4a
are early markers of acute lyme disease in tick bite patients in the Uni-
[6] Ali H et al. Anaphylatoxin C3a receptors in asthma. Resp. Res.
ted States. Int. Arch. Allergy. Immunol. 146:255-261 (2008).
6:19-24 (2005).

[3] Striker RB et al. Complement split products C3a and C4a in

[7] Wills-Karp, M. Complement activation pathways: A bridge
chronic lyme disease. Scan. J. Immunol. 69:64-69 (2008).
between innate and adaptive immune responses in asthma. Proc. Am.
Thorac. Soc. 4:247-251 (2007).
[4] Zhang et al. A complex role for complement in allergic asthma.
Expert Rev. Clin. Immunol. 6(2):269-277 (2010).

20
 4 COMPLEMENT ELISA’S
4.1 OVERVIEW OF AVAILABLE COMPLEMENT ELISA’S

4.2 TECHNICAL INFORMATION ON COMPLEMENT ELISA’S

Pan-Specific C3 Reagent Kit MicroVue™ Quidel®

COMPLEMENT ANIMAL MEASUREMENT IN VIVO AND IN VITRO

Cat. No. 20261

Tests 40
Incubation time 2 hours
Sample volume 20 µl
Sample type Heparin Plasma (10-15 IU/ml Heparin)
Hirudin Plasma (50 μg/ml Hirudin)
Serum
EDTA or citrate plasma cannot be used.
Sample preparation If testing is not to be performed immediately, aliquot samples into appropriate volumes and
store frozen at –70°C
For previously frozen specimens, thaw samples in cold water. After thawing place samples in
ice bath until use.
Species Bovine, Chicken, Dog, Goat, Guinea Pig, Horse, Mini Pig, Pig, Rabbit, Rat, Sheep and Turkey
The Pan-Specific C3 Reagent Kit represents a novel approach to fill the gap of animal-specific
Application Complement ELISAs. The C3 Complement Matrix (CCM) and the C3 Converter Reagent (CCR)
in the kit convert the activity of C3 in the animal specimen to human SC5b-9 that is detectable
with the SC5b-9 Plus EIA kit. Thus the method allows sensitive and quantitative measurement
of C3 in animal plasma or serum, which, to the extent C3 had been consumed prior to the
assay, also provides a measure of prior Complement activation. The method expands the
methodical arsenal of Complement analysis in animals, enabling, among many applications,
npreclinical immune toxicology testing of C-mediated (pseudoallergic) adverse drug effects.

The PS-C3 method is a three-step procedure. In the first step animals are treated with, or
anima sera or plasma samples are incubated with the test drugs or agents or device to
explore possible Complement activation. Samples are collected at appropriate times for the
second step: conversion of animal C3 to human SC5b-9. In the third step SC5b-9 is measured
using the human SC5b-9 Plus ELISA kit (Cat. No. A029).

21 TECOmedical
CIC-C1q MicroVue™ Quidel®
QUANTIFICATION OF CIRCULATING IMMUNE COMPLEXES

Cat. No. A001

Tests 96
Method ELISA
Range 10 - 30 µg Eq/ml
Sensitivity 1.0 µg Eq/ml
Incubation time 2 hours
Sample volume 10 µl (dilute 1:50)
Sample type Serum and plasma

Sample preparation Specimens should be collected aseptically and prepared using standard techniques or
clinical laboratory testing. Do not heat-inactivate the specimens. Sample may be stored at
2-8 ºC for up to 7 days. For longer periods store below -20 ºC.

Reference values One hundred six (106) sera were collected from normal, asymptomatic subjects.
The average CIC concentration was 2.1 µg Eq/mL (S.D.=1.9)

Specifity Complement-CICs are bound to immobilized to C1q-Protein

Species Human, African green monkey, Cynomolgus Macaque, Rhesus macaque, Baboon

Intended use The CIC-C1q Assay is designed for the detection of circulating immune complexes (CIC)
in human serum or plasma. CIC have been measured in a variety of conditions: infections,
autoimmune disorders, trauma and neoplastic proliferative diseases. In addition the
measurement of CIC can be important for the evaluation of certain forms of rheumatoid
arthritis and for monitoring the effectiveness of therapy.
CIC Control
Cat. No. A013
1 Set (2 levels)

22
 CIC-C3d MicroVue™ Quidel®
Raji-Cell-Replacement
QUANTIFICATION OF CIRCULATING IMMUNE COMPLEXES WITH C3 ACTIVATION
FRAGMENTS

Cat. No. A002

Tests 96
Method ELISA
Range 5 - 48 µg Eq/ml
Sensitivity S4 µg Eq/ml
Incubation time 2 hours
Sample volume 10 µl (dilute 1:50)
Sample type Serum and plasma

Sample preparation All specimens should be collected aseptically. Do not heat-inactivate the specimens.
Samples may be stored on ice for up to 6 hours. For longer periods store below -70 ºC.

Reference values Normal ≤

15 µg Eq/ml
Abnormal ≥ 20 µg Eq/ml

Specifity Immobilized monoclonal anti human C3 fragments capture C3d containing immune
complexes.

Species Human

Intended use The CIC-RCR Assay is designed for the detection of circulating immune complexes (CIC) in
human serum or plasma. CIC have been measured in a variety of conditions: infections, au-
toimmune disorders, trauma and neoplastic proliferative diseases. In addition the measure-
ment of CIC can be important for the evaluation of certain forms of rheumatoid arthritis and
for monitoring the effectiveness of therapy.

23 TECOmedical
C1-Inhibitor Plus MicroVue™ Quidel®
FUNCTIONAL C1 INHIBITOR PROTEINS

Cat. No. A037

Tests 96
Method ELISA
Range Concentrations of Functional C1-INH are expressed as Mean Percentage. Range 35 - 100 %.
Incubation time 2 hours
Sample volume 10 µl (dilute 1:101)

Sample type Serum and EDTA plasma

Sample preparation: Specimens should be collected aseptically and prepared. EDTA plasma
sample may be held at room temperature (15-30 ºC) for up to 24 hours. Serum sample should
not be stored at room temperature for longer than 6 hours. If exceeded, the plasma or se-
rum must be stored frozen (-20 ºC or below). Avoid freezing and thawing of the sample.

Reference values Concentrations ≥ 68 % Mean Normal is considered normal.

Specifity C1-INH-Reactant, binds specific to functional active C1-INH.

Species Human, Rhesus macaque, Baboon

Intended use The C1-Inhibitor assay measures the amount of functional C1 inhibitor protein (C1-INH) in
human plasma or serum. This protease inhibitor has enzyme regulating functions. A deficien-
cy of functionally active C1-INH may lead to life-threatening angioedema. Two major forms
of C1-INH deficiency have been reported: the congenital form, termed hereditary angioe-
dema (HAE), and the acquired form, which is associated with a variety of diseases including
lymphoid malignancies. Hereditary angioedema is characterized by transient but recurrent
attacks of nonpruritic swelling of various tissues throughout the body.

24
 iC3b MicroVue™ Quidel®
QUANTIFICATION OF THE IC3B FRAGMENT OF C3 PROTEIN

Cat. No. A006

Tests 96
Method ELISA
Range 0.2 - 2.0 µg/ml
Incubation time 1.5 hours
Sample volume 100 µl (after dilution 1:25 – 1:200)
Sample type Serum and EDTA-plasma

Sample preparation The proper collection and storage of specimens is essential, since C3 is highly susceptible to
proteolysis and hydrolysis. Serum or EDTA plasma specimens should be collected aseptically
using standard techniques. They should be tested immediately or stored at 4 ºC or on ice
until assayed. This should not exceed 4 hours. For long-term storage, freeze at -70 ºC within
2 hours after collection.

Specifity Anti-iC3b monoclonal anti body specifically binds iC3B and not to C3.

Species Human

Intended use The iC3b enzyme immunoassay measures the amount of the iC3b fragment in human plas-
ma or serum, as well as in other biological fluids, experimental samples or mixtures.
The levels of iC3b can be significantly elevated in the serum and plasma of some patients
with complex-associated diseases such as rheumatoid arthritis and systemic lupus erythe-
matodus. iC3b levels may also be elevated in body fluids from other patients with infections,
burns, myocardial infarctions, glomerulonephritis, and acute respiratory distress syndrome.

25 TECOmedical
Bb Plus MicroVue™ Quidel®
BB FRAGMENTS OF FACTOR B OF THE ALTERNATIVE COMPLEMENT PATHWAY

Cat. No. A027

Tests 96
Method ELISA
Range 0.06 - 0.65 µg/ml
Sensitivity LOD: 0.018 µg/ml
LLOQ: 0.033 µg/ml
Incubation time 1.5 hours
Sample volume 100 µl (after dilution 1:10 for plasma, 1:20 for serum)

Sample type Serum and plasma

Sample preparation S pecimens should be tested immediately or stored at 4 ºC or on ice until assayed. This should
not exceed four hours.
For long-term storage, freeze at -70ºC within two hours after collection.

Reference values in Plasma 0.49 - 1.42 µg/ml (+2 SD)

in Serum 0.80 - 6.26 µg/ml (+2 SD)

Specifity Monoclonal mouse-antibody, specifically binds Bb.

Species Human, cynomolgus macaque, baboon, rhesus macaque

Intended use The Bb fragment enzyme immunoassay measures the amount of the complement fragment
of Bb, an activation fragment of Factor B in human plasma or serum, as well as in other
biological fluids, experimental samples or mixtures. This measurement allows a quantitative
assessment of the extent of activation of the alternative pathway of complement in the test
sample. Measurement of alternate pathway activation aids in the diagnosis of several kidney
diseases (e.g. chronic Glomerulonephritis, lupus nephritis), as well as several skin diseases
(e.g. dermatitis herpetiformis and pemphigus vulgaris). Other diseases in which activation of
the alternate pathway of complement has been observed include rheumatoid arthritis, sickle
cell anemia, and gram-negative bacterial infections.

26
 C4a MicroVue™ Quidel®
QUANTIFICATION OF THE C4A FRAGMENT

Cat. No. A036

Tests 96
Method ELISA
Range 5 – 40 ng/ml
Sensitivity LOD: 0.29 ng/ml
LLOQ: 5 ng/ml
ULOQ: 61 ng/ml
Incubation time 2 hours 15 minutes
Sample volume 10 µl (dilute 1:40 for plasma, 1:80 for serum)

Sample type Human and primate plasma or serum, other biological fluids.

Sample preparation Sample collection is critical. Care must be taken to avoid C4a generation in the sample.
For optimal plasma results K2- or K3 EDTA collection tubes are recommended. Serum and
EDTA plasma specimens should be collected aseptically using standard techniques. Samples
should be tested immediately or stored on ice for no longer than 4 hours. For long-term
storage samples should be frozen at -70 ºC, or below.

Reference values Sample n Mean (ng/ml) Range (ng/ml)

EDTA-Plasma 32 1694.65 383.5 – 8168.17

Serum 44 1098.00 20.92 – 4437.24

Specifity Monoclonal mouse-antibody, specifically binds human C4a and C4a-des Arg.

Species Human, primate

Intended use The C4a Enzyme Immunoassay Kit measures the amount of the complement fragment C4a,
an activation fragment of complement protein C4 in human and primate plasma, serum and
other biological fluids. Measurement of C4a in human plasma or serum provides evidence for
the involvement of the classical or lectin pathway of complement.
Under normal conditions, activation of the classical or lectin complement pathways results
in the cleavage of the complement protein C4 into C4a and C4b by the protease C1s. C4a is
rapidly cleaved to its more stable, less active form C4a-des Arg by endogenous serum car-
boxypeptidase N enzyme. Thus quantitation of C4a (C4a plus C4a-des Arg) should provide a
reliable measurement of classical or lectin pathway activation that has occurred in the test
samples.

• Rheumatoid Arthritis
• Systemic Lupus Erythematosis (SLE)
• Lyme Disease

27 TECOmedical
C4d MicroVue™ Quidel®
QUANTIFICATION OF C4D-CONTAINING FRAGMENTS OF ACTIVATED C4 OF THE
CLASSICAL COMPLEMENT PATHWAY

Cat. No. A008

Tests 96
Method ELISA
Range 0.025 - 0.25 µg/ml
Sensitivity LOD: 0.01 µg/ml
LLOQ: 0.022 µg/ml
Incubation time 1.5 hours
Sample volume 10 µl (dilute 1:70 for normal Samples)
Sample type Serum, EDTA plasma or other biological fluids

Sample preparation The proper collection and storage of specimens is essential, since C4d is highly susceptible to
proteolysis. Serum or EDTA plasma specimens should be collected aseptically using standard
techniques. They should be tested immediately or stored at 4 ºC or on ice until assayed. This
should not exceed four hours. For long-term storage freeze at -70 ºC within two hours after
collection.

Reference values EDTA plasma 0.7 µg – 6.3 µg/ml (+2SD)

Serum 1.2 µg – 8.0 µg/ml (+2SD)

Specifity Monoclonal mouse-antibody, specifically binds C4d.

Species Human, cynomolgus macaque, baboon, rhesus macaque

Intended use The C4d fragment enzyme immunoassay measures the amount of the C4d-containing
activation fragments of C4 (C4b, iC4b, and C4d) present in human serum, EDTA plasma and
other biological or experimental samples.
The levels of C4d, when normalized for the presence of endogenous C4, can be significant-
ly elevated is plasma specimens obtained from some patients with rheumatoid arthritis,
hereditary angioedema, systemic lupus erythematosis and other illnesses. Cd4 may also be
elevated in body fluids and plasma samples obtained from patients in which classical
complement pathway activation is known to occur, e.g. from patients with a variety of
humoral autoimmune diseases, septicemia, thermal injury, multiple organ trauma, myocar-
dial infarction, hereditary angioedema, glomerulonephritis and acute respiratory distress
syndrome.

28
 SC5b-9 Plus MicroVue™ Quidel®
QUANTIFICATION OF THE SC5B-9 COMPLEX

Cat. No. A029

Tests 96
Method ELISA
Range 10 -170 ng/ml
Sensitivity LOD: 3.7 ng/ml
LLOQ: 8.8 ng/ml
Incubation time 2 hours
Sample volume 50 µl (dilute 1:40 for serum)
10 µl (dilute 1:10 for plasma)
Sample type Serum, EDTA plasma, spinal fluid or other biological fluids

Sample preparation The proper collection and storage of specimens is essential since SC5b-9 may be generated
in improperly handled specimens. Serum or EDTA plasma specimens should be collected
aseptically using standard techniques. They should be tested immediately or stored at 4 ºC
or on ice until assayed. This should not exceed four hours. For longer-term storage freeze at
-70 ºC. Plasma concentrations better reflect in vivo concentrations in comparison to serum
concentrations.

Reference values Serum 334 - 1672 ng/ml

EDTA Plasma 127 - 303 ng/ml

Specifity Monoclonal mouse-antibody, specifically binds SC5b-9.

Species uman, African green monkey, cynomolgus macaque, baboon, rhesus macaque, Pigtail
H
monkey

Intended use The SC5b-9 enzyme immunoassay measures the amount of SC5b-9 present in human
plasma, serum and other biological or experimental samples.
The Terminal Complement Complex (TCC, SC5b-9) is generated by the assembly of
C5 through C9 as a consequence of activation of the complement system by either the
classical, lectin or alternative pathway. The membrane attack complex (MAC), a form of TCC,
is a stable complex that mediates the irreversible target cell membrane damage associated
with complement activation.

29 TECOmedical
C3a Plus MicroVue™ Quidel®
QUANTIFICATION OF THE C3A FRAGMENT

Cat. No. A032

Tests 96
Method ELISA
Range 0.05 – 5 ng/ml
Sensitivity LOD: 0.012 ng/ml
0.023 ng/ml
Incubation time 2 hours 15 minutes
Sample volume 10 µl (dilute 1:200 for plasma, 1:5000 for serum)

Sample type Human plasma, serum or other biological fluids.

Sample preparation Sample collection is critical. Care must be taken to avoid C3a generation in the sample. For
plasma, blood samples should be collected with disodium EDTA as anticoagulant and should
be centrifuged at 2000xg at 2-8 °C.
The entire operation must be completed immediately. Samples should be prepared and as-
sayed immediately or stored on ice for up to 2 hours.
For long-term storage freeze at -70 ºC with stabilizing solution.

Reference values Serum 71.0 – 589.2 ng/ml

EDTA Plasma 33.8 – 268.1 ng/ml

Specifity Monoclonal mouse-antibody, specifically binds C3a-desArg.

Species Human, African green monkey, cynomolgus macaque, baboon, rhesus macaque

Intended use The C3a enzyme immunoassay measures the amount of C3a-desArg in human EDTA plasma,
serum and other research samples.
Under normal conditions, activation of the classical, alternative or lectin complement
pathways results in the formation of a C3 convertase muli-molecular enzyme capable of
cleaving C3 to C3a and C3b. C3a is a low molecular weight (approximately 9 kD) protein frag-
ment of 77 amino acids. C3a is rapidly metabolized by the serum enzyme, carboxypeptidase
N, to the more stable, 76 amino acid form, C3a des-Arg.
The quantitation of C3a des-Arg therefore provides a reliable measurement of the level of
complement activation in the test sample.

30
 C5a MicroVue™ Quidel®
MEASUREMENT OF TERMINAL COMPLEMENT PATHWAY ACTIVATION IN EXPERIMENTAL
SAMPLES

Cat. No. A025

Tests 96
Method ELISA
Range 0.1 - 1 ng/ml
Sensitivity LOD: 0.01 ng/ml
LLOQ: 0.050 ng/ml
Incubation time 2 hours 15 minutes
Sample volume 20 µl (dilute 1:20 for plasma)
10 µl (dilute 1:50 for serum)
Sample type Serum, EDTA- and citrated plasma.

Sample preparation The proper collection, storage and shipment of specimens are essential. Test immediately
or stored up to 4 hours at 2-8 ºC or on ice. For long-term storage the samples should kept
frozen at -70 ºC with stabilizing solution (Cat. No. A9576), dilute samples 1:1 with stabilizing
solution.

Reference values EDTA Plasma 0.37 – 74.33 ng/ml

Serum 13.37 – 179.23 ng/ml

Specifity C5a and C5a des-Arg

Species Human

Intended use C5a is generated as a result of cleavage of the terminal complement protein C5,during
activation of the complement system via the classical, alternative or lectin pathway. C5a is
a low molecular weight (approximately 9 kD) protein fragment of 74 amino acids. C5 a is
rapidly metabolized by the serum enzyme carboxypeptidase to more stable, less active, 73
amino acid form, C5a des-Arg.

Research has associated elevated levels of fluid phase and adsorbed C5a with hemo-incom-
patibility of some biomaterials, particularly in extracorporeal circuits. Levels of C5a have also
been associated with pathogenesis of a variety of disease states, including myocardial infarc-
tion, stroke, as well as vascular leak syndrome and associated kidney injury. The role of C5a
in the pathogenesis of malaria and other infectious diseases, as well as sepsis, is likewise well
documented.

31 TECOmedical
CH50 Eq MicroVue™ Quidel®
TOTAL CLASSICAL COMPLEMENT PATHWAY ACTIVITY

Cat. No. A018

Tests 96
Method ELISA
Range Appr. 0 - 300 U Eq/ml
Incubation time 3.5 hours
Sample volume 14 µl (dilute 1:200)
Sample type Serum ONLY. Plasma CANNOT be used.

Sample preparation The proper collection, storage and shipment of specimens are essential, since complement
may be activated in improperly handled specimens. Assay immediately or keep on ice for
testing within 4 hours, up to 3 days at 4°C. For long-term storage, freeze at -70 ºC. Maximum
6 freeze/thaw cycles.

Reference values 133 ± 54 U Eq/ml

Specifity The monoclonal antibody specific to terminal complement complexes arising as the result of
the activation step in the test.

Species Human, cynomolgus macaque

Intended use The binding of C1q component of C1 to immune complexes triggers the classical comple-
ment pathway. This activation results in a cascade of enzymatic and non-enzymatic reacti-
ons, culminating in the formation of terminal complement complexes (TCC). Under standard
conditions, the level of TCC that can be generated in serum is a quantitative expression of
the serum’s total classical complement activity. The MicroVue CH50 Eq EIA is designed to
measure the total classical complement pathway activity in human serum samples. The mea-
surement of CH50 allows detection of deficiencies of one or more complement components
(C1 through C9).

32
 Ba MicroVue™ Quidel®
QUANTIFICATION OF THE COMPLEMENT BA FRAGMENT

Cat. No. A034

Tests 96 ELISA
Method 0.05 – 2.1 ng/ml
Range LOD: 0.011 ng/ml
Sensitivity LLOQ: 0.033 ng/ml
Incubation time 3 hours
Sample volume 10 µl (dilute plasma 1:1000, serum 1:2000), 25 µl urine (dilute 1:15)
Sample type EDTA Plasma, serum, urine
Sample preparation Serum / Plasma: The Ba fragment of Factor B is susceptible to proteolysis. For optimal plasma
results, K2 EDTA should be used. Collect blood sample and centrifuge immediately at 2-8°C.
Assay immediately, do not store longer than 2 hours at 2-8°C. For longer storage -70°C.
Urine: Collect preservative-free first Morning void (FMV) or second morning void (SMV)
before 10:00 am. Store sample refrigerated (2-8°C) for less than 1 day, or freeze the sample at
-70°C for longer storage. Maximum 5 freeze and thaw cycles.

Reference values Sample n Mean (ng/ml) Range (ng/ml)

EDTA-Plasma 35 658 226 – 2153

Serum 29 1642 436 – 3362

Urine 167 7.7 0.6 – 27

Specifity Monoclonal mouse-antibody, specifically to capture the Ba fragment.

Species Human, African green monkey, cynomolgus monkey, rhesus monkey, canine

Intended use By quantifying the amount of Ba, the extent of alternative pathway activation at the time of
sample collection can be determined.

Activation of the alternative pathway has been associated with a variety of disease states
including SLE, chronic glomerulonephritis, rheumatoid arthritis, sickle cell anaemia and gram
negative bacterial infections.

The activation of the alternative complement pathway can be triggered by a variety of

substances including microbial polysaccharides or lipids, gram-negative bacterial lipopolys-
accharides, surface determinants present on some viruses, parasites, virally infected mam-
malian cells, and cancer cells. In autoimmune
diseases, the alternative complement pathway may contribute directly to tissue damage.
Alternative complement pathway activation may also be an indicator of haemo-incompati-
bility of biomaterials.

Application • kidney diseases • pemphigus vulgaris

• chronic glomerulonephritis • age-related macular degeneration
• lupus nephritis • fetal loss in at risk pregnancy
• skin diseases • rheumatoid arthritis
• dermatitis herpetiformis • sickle cell anaemia

33 TECOmedical
 8.1.1CROSS
4.3 Crossreactivity of complement
REACTIVITY OF fragment
COMPLEMENT ELISA’S ELISA’s SAMPLES
TO PRIMATE
to primate samples
Summary of Results:
Species crossreactivity of various primate plasma and sera were assessed in the Quidel® MicroVue™ Complement Pro-
duct ElA‘s as well as the Quidel® MicroVue™ CH50 Eq EIA kit. Of the assays tested, C3a, Bb, C4d, SC5b-9 and CH50 all
demonstrated a high degree of species crossreactivity with baboon and old world monkey samples . Results for normal
and activated sera were comparable to human control samples .

Experiment I:
Samples were tested for the ability to block activated human complement binding to the neo-antigen specific monoclo-
nal capture antibodies via ELISA inhibition assay . The capture monoclonals are the same antibodies used in the Quidel®
MicroVue™ Complement Product assays . Positive blocking ability was defined as greater than 80 % reduction in signal
vs . a standard dilution of activated human serum giving endpoint signals (OD 405) between 1 .2 and 1 .4 units in the assay .
Results are shown in Table I .

African
Cynomologus Marmo- Squirrel
Baboon Rhesus lemur Green Canine Porcine
Macaque set Monkey
Monkey
Ba A034 - + + N/T N/T N/T + + -
Bb A027 + + + - - - - N/T N/T
iC3b A006 N/T - - - - - - - -
SC5b-9 Plus A029 + + + - - - - - -
C4d A008 + + + - - - - - -
C3a A032 + + + N/T N/T N/T - N/T -
C5a A025 - - - N/T N/T N/T - - -

Figure 19:
EIA Inhibition Results

+ Crossreactivity
N/T Not Tested
- No crossreactivity

Experiment II:
Species crossreactivity in the ELISA assays was shown using various complement activating substances (Cobra Venom
Factor, Zymosan and Heat Aggregated Gamma Globulin) .These products activate specific proteins and complement
pathways . Normal results are shown in Table II . Positive cross-reactivity was defined as a tenfold or greater increase in
fragment levels after activation . This threshold was based upon comparable human responses . Of the assays tested, clear
cross-reactivity was shown for cynomologus, baboon and rhesus monkey samples in Bb, C4d, C3a, SC5b-9 and CH50
EIA kits . No specific reaction could be demonstrated using marmoset sera or any non-human sera in the IC3b EIA .

Bb (µg/ml) C4D (µg/ml) iC3b (µg/ml) C3a (ng/ml) SC5b-9 (ng/ml) CH50 (U Eq/ml)

Human (N = 5) 1 .5 ± 0 .975 4 .1251 ± 2 .55 51 .0 ± 1 .5 1209 ± 177 237 ± 28 110 .3 ± 6 .3

Cynomologous
0 .83 ± 0 .45 4 .065 ± 0 .82 NR 879 ± 110 241 ± 28 72 .9 ± 13
Macaque (N = 10)
Baboon (N = 12) 0 .75 ± 0 .43 3 .75 ± 1 .5 NR 900 ± 177 165 ± 57 .5 NT
Marmoset (N = 8) NR 4 .41 ± 1 .8 NR NR NR NR

Figure 20:
Crossreactivity of selected primate sera with the CSP assay kits

38
34
 Selected References

[1] Birmingham D, Lee HA et al . (1999)

Effects of Immune complex formation and
complement activation on circulating platelets
in the primate.
Clin Immuno 91, 1 .

[2] Henry SP, Giclas PC et al . (1997)

Activation of the alternative pathway of
complement by a phophorothioate
oligonucleotide: Potential mechanism for action.
J Pharm Exp Thera 281, 2810 – 2816 .

[3] Katopodis AG, Warner RG et al . (2002)

Complement analysis in the 21st century.
Mol . Immunol ., Sep, 44 (16): 3838 – 49 . Review

[4] Kirshfink M (1998)

The Clinical Laboratory: Testing the Complement
System. In: The Complement System.
Rother K and Till GO (eds), Springer Verlag Berlin
Heidelberg, pp . 522 – 547 .

[5] Loss M, Vangerow B, et al . (2000)

Acute vascular rejection is associated with
systemic complement activation in a
pig-to-primate kidney xenograft model.
Xenotransplantation 7, 186 – 196 .

[6] Mocco J, Tanvir C (2002)

HuEP5C7 as a humanized monoclonal anti- E/P
selectin neurovascular protective strategy in a
blinded placebo controlled trial of nonhuman
primate stroke.
Circ Res 91, 907 – 914 .

[7] Mollnes TE (1997)

Analysis of in vivo complement activation.
In: Complement and Complement Receptors.
Weir (ed), pp . 78 .1 – 78 .6 .

[8] Undar A, Eichststaedt HC et al . (2002)

Novel anti-factor D monoclonal antibody
inhibits complement and leukocyte activation in
a baboon model of cardiopulmonary bypass.
Ann Thorac Surg 74, 355 – 362 .

39

35 TECOmedical
 5 COMPLEMENT ANTIBODIES AND REAGENTS
9. Complement antibodies and reagents
Depleted or Deficient Sera Purified Proteins

Except for the C3-dpl, C3/C4-dpl and the C4-deficient gui- Each complement protein has been tested for functional pu-
nea pig sera, a specific complement protein has been remo- rity in standard hemolytic assays and for biochemical purity
ved immunochemically from each depleted human serum by SDS-polyacrylamide gel electrophoresis . The concentra-
reagent . Depleted sera are well suited for the detection and tion of each complement protein, except for Factor D and
quantitation of hemolytically active complement proteins . C3a is approximately 1 .0 mg/ml . Volume: 250 µl per vial .
Except for the specifically depleted component, the classi-
cal and alternative pathways are intact . 1 .0 ml per vial . Catalog No .
C1q A400 (1 .0 ml)
Catalog No .
C3 A401
C2 Depleted A500
C4 A402
C5 Depleted A501
C5 A403
C6 Depleted A502
C6 A404
C7 Depleted A503
C7 A405
C8 Depleted A504
C8 A406
C9 Depleted A505
C9 A407
Factor B Depleted A506
Factor B A408
C4 Depleted (Guinea Pig) A507
Factor D A409
C3 Depleted A508
Factor H A410
C1Q Depleted A509
Factor I A411
Factor P Depleted A512
Factor P A412
C3/C4 Depleted A521
C3b A413 (50 µl)
IgG Depleted
C3a A414 (100 µl)

Complement Reagents
Chicken Polyclonal Antibodies (IgY)
Catalog No .
Each polyclonal IgY antibody has been isolated from
CVF, Cobra Venom Factor A600 (1 .0 ml)
chicken egg yolks and tested for purity by SDS-poly-
Human Complement Standard A100 (1 .0 ml) acrylamide gel electrophoresis . Specificity and activity were
assessed by EIA . The protein concentration of each is ap-
Normal Guinea Pig Serum A119 (1 .0 ml)
proximately 1 mg/ml in phosphate buffered saline . Volume:
Complement
100 µl per vial .
Normal Human Serum
A112 (2 .5 ml)
Complement Catalog No .
Normal Human Serum Anti Human C3d A800
A113 (5 .0 ml)
Complement
Anti Human C4b A801
Complement Activator (HAGG) A114 (0 .2 ml)
Anti Human SC5b-9 A802
Complement Sample Panel A115 (10 x 0 .025 ml)
CVF Activated-NHS A116 (0 .1 ml)
CIC Sample Panel A117 (5 Samples)
Specimen Stabilizing Solution A9576 (25 ml)

36
40
 Monoclonal antibodies Biotinylated Monoclonal Antibodies

Each monoclonal antibody has been purified from mouse Biotin labeled Monoclonal Antibodies
ascites fluid and tested for purity by SDS- polyacrylamide Each monoclonal antibody has been purified from ascites
gel electrophoresis . The protein concentration of each is 1 fluid and labeled with biotin . The protein concentration of
mg/ml in borate buffered saline with 0 .02 % sodium azide . each is 0 .2 mg/ml . Volume: 250 µl per vial .
Volume: 100 µl per vial .
Catalog No .
Catalog No .
C1q A700
C1q A201
C3c A701
C3a A203
C3d A702
C3c A205
C4c A703
C3d A207
C4d A704
iC3b (Neoantigen) A209
C5 A705
C4c A211
C6 A706
C4d A213
C7 A707
C4 Binding Protein A215
C8 A708
C5 A217
C9 A709
C6 A219
iC3b (neoantigen) A710
C7 A221
SC5b-9 (neoantigen) A711
C8 A249
Factor Bb A712
C9 A223
Factor B (Ba ) A225
C3 Proactivator Goat Polyclonal Antisera
Factor B (Bb) A227 Quidel’s complement antisera are raised in goats and are
C3 Proactivator
quality controlled for specificity by immunochemical ana-
Factor H #1 A229 lysis . Each contains 0 .02 % sodium azide . Volume: 2 .0 ml
per vial .
Factor I #1 A247
Factor I #2 A231 Catalog No .
Factor P #1 (Properdin) A233 C1 Inhibitor A300
Factor P #2 (Properdin) A235 C1q A301
S Protein (Vitronectin) A237 C1s A302
SC5b-9 A239 C2 (Ig Fraction) A303 1 .0 ml
(TCC-Neoantigen)
C3 A304
Clusterin A241
C4 A305
(SP40, 40 und APO J)
C3d (Neoantigen) A250 C5 A306

C4d (Neoantigen) A251 C6 A307

Bb (Neoantigen) A252 0 .5 ml C7 A308

C4d (Neoantigen) A253 C8 A309

Factor H #2 A254 C9 A310

Factor H #3 A255 Factor B A311

Factor H A312
Factor I A313

37 TECOmedical
41
 10. Complement Specific Antisera: Application Overview

Preparation

Hyperimmune antisera were raised in goats by standard immunization procedures utilizing highly pure complement
antigens isolated from freshly drawn normal human plasma . Antiserum absorption, if required, was accomplished by
solid phase adsorption procedures . The antisera were judged to be monospecific when tested at various concentra-
tions against freshly drawn normal human serum containing 10 mM EDTA by double immunodiffusion, one and two
dimensional immunoelectrophoresis and rocket immunoelectrophoresis . Antisera were dilapidated and contain
0 .02 % sodium azide as a preservative .

Applications

Quidel’s complement antisera have been used in a variety of techniques including double immunodiffusion, quantitative
radial immunodiffusion (RID), rocket immunoelectrophoresis, western blotting, immunohistochemistry and ELISA . It is
important to note that these products are whole antiserum and therefore may require appropriate whole serum controls
in some applications .

Double Immunodiffusion

Optimal dilutions vary by application . In general, Quidel’s antisera will provide clear precipitin lines at a 1:8 or greater
dilution under the following conditions:
• Agarose: 1.5 % agarose in 25mM barbital buffer (pH 8.6), 125 mM NaCl, 10 mM EDTA
• Well size: 4 mm in diameter
• Sample: 18 μl of undiluted NHS.
• Pattern developed for 24 hours at room temperature.
Antigen or antibody excess may yield “fuzzy” precipitin lines .

Quantitative Radial Immunodiffusion

Recommended antiserum concentrations are between 0 .5 and 3 .0 µl/cm2 under the following conditions .
• Agarose: 1.5 % agarose in 25 mM barbital buffer (pH 8.6), 125 mM NaCl, 10 mM EDTA
• Well size: 3mm in diameter
• Sample size: 10 μl of each serum dilution
• Patterns developed for 48 to 96 hours in a moisture chamber at RT
If a greater range of NHS dilutions are required for a particular application, the amount of antiserum (µl/cm2) should be
decreased by half . Precipitin rings should be visualized after staining .

Item Antigen Input of antiserum Optimal dilution

A300 C1 Inhibitor 1 .5 µl/cm2 1:2
A301 C1q 0 .8 µl/cm2 1:2
A302 C1s 2 .5 µl/cm2
1:2
A303 C2 N/T N/T
A304 C3 0 .8 µl/cm2 1:4
A305 C4 2 .5 µl/cm2 1:4
A306 C5 1 .0 µl/cm2 1:2
A307 C6 1 .2 µl/cm2 1:2
A308 C7 3 .0 µl/cm2 1:2
A309 C8 3 .0 µl/cm2 1:2
A310 C9 1 .5 µl/cm2
1:2
A311 Factor B 0 .8 µl/cm2
1:2
A312 Factor H 1 .0 µl/cm2 1:2
A313 Factor I 1 .5 µl/cm2 1:2

Figure 21:
Specific results for RID

42

38
 Table continues on next page >>

10.1PRODUCT
5.1 Product INFORMATION
Information Monoclonal
MONOCLONAL Antibodies
ANTIBODIES
Item Number Description (murine anti human) Epitope description Isotype
A201 C1q Binds to globular head of C1q IgG1k

A203 C3a Binds C3 and C3a IgG1k

A205 C3c Binds C3 and C3c IgG1k

A207 C3d Binds C3 and C3d containing fragments IgG1k

A209 iC3b Neo antigen, binds only iC3b IgG2bk

A211 C4c Binds C4 and C4c containing fragments IgG1k

A213 C4d Binds C4 and C4d containing fragments IgG1k

A215 C4 Binding Protein IgG2bk

A217 C5 Inhibits lysis of Sheep EA, does not IgG1k
recognize MAC bound to membrane
A219 C6 Recognizes MAC IgG1k
A221 C7 Inhibits lysis of Sheep EA, does not IgG1k
recognize MAC bound to membrane
A249 C8 Recognizes MAC IgG2ak
C9 Does not inhibit lysis of sheep EA, does
A223 IgG2bk
not recognize MAC
A225 Factor B (Ba) Inhibits function of Factor B; Binds both IgG1k
C3 Proactivator nascent Factor B and the Ba cleavage
product
A227 Factor B (Bb) C3 Proactivator Inhibits function of Factor B; Binds both IgG1k
nascent Factor B and the Bb cleavage
product
A229 Factor H #1 Does not inhibit the function, Factor H
IgG1k
like Protein is not detected
A247 Factor I #1 Does not inhibit the function IgG1k
A231 Factor I #2 Does not inhibit function of Factor I IgG1k
A233 Factor P #1 (Properdin) Blocks Function IgG1k
A235 Factor P #2 (Properdin Does not inhibit function of Factor P IgG1k
A237 S Protein (Vitronectin) Binds free S protein (Vitronectin) as well IgG1k
as SC5b-9
A239 SC5B-9 Directed against a neo antigen on poly IgG2ak
C9 and denatured C9
A241 Clusterin Clusterin is also called Apo J and IgG1k
SP40,40
A250 C3d (Neoantigen) Binds to C3d containing activation
products of C3 Only
A251 C4d (Neoantigen) Binds C4d activation products of C4
only
A252 Bb (Neoantigen) Binds the Bb Sub Unit Only

A253 C4d (Neoantigen) Binds C4 and C4d containing fragments IgG1k

A254 Factor H #2 Does not inhibit the function lgG1k

A255 Factor H #3 Does not inhibit the function lgG1k

Notes:
All Complement Monoclonals are provided in 100 μl fill volumes at 1.0 mg/ml in Borate Buffered Saline. Suggested initial dilution for all antibodies in IHC
applications is 1:1000; actual application and tissue type will determine proper dilution.

39 TECOmedical

44
 Tested Applications
ELISA RIA IHC W .Blot FACS Function Notes Dilution ELISA
X X N/T N/T X Probably does not inhibit function of C1q molecule 1:16000
C3a does not bind to cells so is not usually detected in FACS
X X N/A X N/A
r IHC
Binds both the fragment and whole protein so is not suitable
X X X X X 1:6200
for all EIA applications
Binds both the fragment and whole protein so is not suitable
X X X X X 1:10500
for all EIA applications
Only reactive to the activation product iC3b, not to C3b or
X X X X X
other products
Binds both the fragment and whole protein so is not suitable
X X X X X 1:5400
for all EIA applications
Binds both the fragment and whole protein so is not suitable
X X X X X 1:64000
for all EIA applications
X N/T N/T N/T N/T

X X N/A X N/A 1:10000

X X X N/T X

X X N/A X N/A

X N/T X X X

X X N/A N/T N/A

Binds both the fragment and whole protein so is not suitable

X X X X X
for all EIA applications

Binds both the fragment and whole protein so is not suitable

X X X X X
for all EIA applications

X N/T X X X > 1:1000

X X X X N/T 1:34000
X X X X N/T
X X X N/T N/T
X X X N/T N/T

X X X X X

X X X X X

X X X X X

X N/T X

X N/T X

X N/T X
Binds both the fragment and whole protein so is not suitable
X X X X X
for all EIA applications
X X X X N/T > 1 µg/ml
X X X N/T N/T > 1 µg/ml

For IHC the ABC method has proven to be the most reliable overall for these antibodies and analytes; frozen tissue is preferred. Antibodies indicated in
bold are used in the EIA kits from QUIDEL. Suggested initial dilution for all antibodies in Westernblot 10 ug/ml.
For FACS the recommended dilution is 10-40 ug/ml – 1:100 dilution – app 200 samples per vial.

40 45
 Immunohistochemistry

The optimal dilutions of antibodies vary with the specific tissue type and conditions of the experiment . In general,
polyclonal and monoclonal antisera from QUIDEL have optimal dilutions between 1:1000 and 1:50000 in histolo-
gical applications . We strongly suggest that, when optimizing the IHC experiment, this range is bracketed to ensure
detection of the proper optimal dilution .
Several techniques for histological evaluation of complement deposition have been published using QUIDEL’s mono-clonal
antibodies to complement antigens [1 – 4] . The following protocol (adapted from Rogers, et al .) describes one method
that has provided excellent results in several studies . Experimental procedures should take into consideration the type
of sample, conditions under which the sample was obtained, the specific antibodies and secondary reagents and other
lab-specific pheno-menon . This protocol is provided for reference use only .

Tissue Preservation

Tissue samples were fixed in 4 % (wt/vol) paraformaldehyde (0 .1 M sodium phosphate buffer, pH 7 .4) for 24 hours and
then cryoprotected in 2 % DMSO/10 % glycerol followed by 2 % DMSO/20 % glycerol for 48 hours each . Samples were
sectioned on a freezing microtome at 20 µm and stored at -20 ˚C in a solution of 33 % (vol/vol) glycerol, 33 % polyethylene
glycol and 33 % phosphate buffer (0 .1M, pH 7 .4) .

Histology

Sections were washed 6 x for 15 minutes each in TBS (0 .01 M Tris-HCl, pH 7 .6/0 .09 M NaCl) . Endogenous peroxidase
was blocked by incubation with 0 .3 % H202 in 50 % methanol (vol/vol) in TBS followed by 3 x 15 minute washings with
TBS/0 .5 % Triton X 100 . Samples were blocked with 3 % BSA for one hour . Sections were incubated overnight at 4 °C with
the optimal dilution of primary antibody (optimal was determined by incubating serial dilutions of antibody from 1:500 to
1:128000 on a strong positive control tissue) . Sections were then washed 3 x with TBS/0 .05 % Triton X-100 and incubated
with biotin conjugated secondary antibody as per the manufacture’s instructions . The bound secondary anti-body was
allowed to react with DAB (diaminobenzidene) as in standard ABC/DAB
immunohistochemistry .

Controls

Suggested negative controls include omission of the primary antibody, pre-absorption with the purified comple-
ment antigen and standard negative tissue . Suggested positive controls include disease state kidney samples
(glomeruonephritis) and Alzheimer’s brain .

ELISA

Optimal dilutions for these antisera are greater than 1:50,000 in an antigen capture ELISA . Specific optimal dilutions vary
by lot and assay type .

General Notes

In the case of the QUIDEL polyclonal antisera (A300’s), blocking with an appropriate dilution of normal goat sera is
suggested .

Selected References

[1] Rogers, J ., Cooper, N . et al . (1992) [3] Baldwin, W . et al . (1999)

Complement Activation by β-amyloid in Complement deposition in early cardiac
Alzheimer disease. transplant biopsies is associated with ischemic
PNAS 89:10016-10020 injury and subsequent rejection episodes.
Transplantation 68:6
[2] Qian, Z . Wasowska, B .A ., Baldwin, W ., et al . (1999)
C6 produced by macrophages contributes to [4] Murphy, B, Kirszbaum, L, et al . (1998)
cardiac allograft rejection. SP-40,40, a newly identified normal human serum
Amer . J . Path 155:4 protein found in SC5b-9 complex of complement
and in immune deposits in glomerulonephritis.
Clin Invest 81:1858 – 1864k

41 TECOmedical
43
 5.2
10.2ANIMAL
AnimalSPECIES
SpeciesCROSSREACTIVITY
CrossreactivityMONOCLONAL ANTIBODIES*
Monoclonal Antibodies*
Species Crossreactivity Monoclonal Antibodies*

ANTI-HUMAN ANTIBODY CROSS-REACTING SPECIES

GUINEA PIG

HAMSTER
CHICKEN

MONKEY
BABOON

HUMAN

MOUSE

RABBIT
HORSE

SHEEP
COW

DOG
CAT

RAT
PIG
C1q A201 - - - - - - - - + slight - N/T - - -
C3a A203 + - - - - - - - + + - + - - -
iC3b (neo) A209 - - - - - - - - + N/T - N/T - - -
C3c A205 + + - - - + - + + + - N/T - - -
C3d A207 - - - - - - - - + - - - - - -
C4c A211 + - - - - - - - + + - N/T - - -
C4d (neo) A251 + N/T N/T N/T N/T N/T N/T N/T + + N/T N/T N/T N/T N/T
C4/C4d A213 - - - - - - - - + - - - - - -
C5 A217 + - - - - - - - + + - N/T - - -
C6 A219 N/T N/T N/T N/T N/T N/T N/T N/T + N/T N/T N/T N/T N/T N/T
C7 A221 - + - + + - + + + N/T - + + + -
C8 A249 N/T N/T N/T N/T N/T N/T N/T N/T + N/T N/T N/T N/T N/T N/T
C9 A223 + - + + - + + + + + - N/T - - -
SC5b-9 (TCC)(neo) A239 + - - - - - - - + + - N/T - - -
Factor H (#1) A229 + - - - - - - + + + - N/T - - -
Factor H (#2) A254 + - - - - - - - + + - N/T - - -
Factor H (#3) A255 + - - - - - - - + + - N/T - - -
Factor I (#1) A247 - - - - - - - - + N/T - N/T - - -
S Protein A237 - - - - - - - - + N/T - N/T - - -
Clusterin/SP40,40 A241 N/T N/T N/T N/T N/T N/T N/T N/T + + - N/T N/T - N/T

+ Crossreactivity
| Crossreactivity by Immunohistochemistry**
N/T Not Tested
- No crossreactivity

*Monoclonal antibodies were tested at QUIDEL for species crossreactivity in ELISA inhibition assays .
A more sensitive assay may detect cross reactivity where none has been indicated by ELISA inhibition .

**Immunohistochemistry data are compiled from the observations of outside researchers;

these experiments were not performed at QUIDEL and have not been confirmed .

42

46
 5.3 ANIMAL
10.3 Animal SPECIES
SpeciesCROSSREACTIVITY
Crossreactivity POLYCLONAL ANTIBODIES
Polyclonal Antibodies
Species Crossreactivity Polyclonal Antisera

CROSS-REACTING SPECIES

GUINEA PIG

HAMSTER
CHICKEN
BABOON

HUMAN

MOUSE

RABBIT
HORSE

SHEEP

GOAT
COW

DOG
CAT

RAT
PIG
Goat anti-human C1-INH A300 +++ - - - + - - - +++ - - + - - -
Goat anti-human C1q A301 +++ +++ - - +++ +++ +++ + +++ +++ - +++ +++ - -
Goat anti-human C1s A302 +++ - - - - - - - +++ - - - - - -
Goat anti-human C3 A304 +++ - - - - +++ +++ +++ +++ +++ + +++ +++ - -
Goat anti-human C4 A305 +++ + - - +++ - +++ +++ +++ + + +++ +++ - -
Goat anti-human C5 A306 +++ + - - +++ +++ + +++ +++ + +++ +++ +++ - -
Goat anti-human C6 A307 +++ +++ - - +++ - - + +++ - - +++ +++ - -
Goat anti-human C7 A308 +++ +++ - - - +++ + + +++ - - +++ - - -
Goat anti-human C8 A309 +++ + - - +++ +++ + + +++ + - +++ + - -
Goat anti-human C9 A310 +++ - - - - - + +++ +++ - - + - - -
Goat anti-human Factor B A311 +++ +++ - - - +++ +++ +++ +++ +++ - +++ +++ - -
Goat anti-human Factor H A312 +++ +++ - - +++ + +++ +++ +++ +++ - +++ +++ - -
Goat anti-human Factor I A313 +++ - - - - - - - +++ - - - - - -

+++ Strong cross-reaction

++ Moderate cross-reaction
+ Weak cross-reaction
- No cross-reaction
NT Not Tested

All antisera were tested for species crossreactivity by double

immunodiffusion and/or one-dimensional immunoelectrophoresis.

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