Principles of Antigen-Antibody Reactions
Law of Mass Action
Mechanism of Antigen-Antibody Reactions:
1. Primary Union of Antigens and Antibody
A. Lock and Key-Ag and Fab of Ab must be
complementary to one another
*X-ray crystallography studies: Antigenic
determinant nestles in a cleft formed by the
combining site of the antibody
*Ag-AB reactions is one of a key (i.e. the Ag)
which fits into a lock (i.e. the Ab)
*the lock and key relationship between
epitope and paratope is enhanced by induced
fit due to their mutually deformable
conformations
B. Non-Covalent Bonds
-Bonds:
 Hydrogen
 Ionic
 Hydrophobic Interactions
 Van der Waals Forces
-Each bond is weak; many are strong
-To “hold” they must be close --- requiring
high amounts of complementarity
C. Reversibility-Since Ag-Ab reactions occurs via
non-covalent bonds they are by their nature
reversible
2. Affinity
-Affinity of an antigen-binding site for an antigen
-the association constant between antibody and a
univalent antigen
-the strength of the reaction between a single
antigenic determinant and a single combining site
on the antibody
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3.
-most antibodies have a high affinity for their
antigens
Ag+ Ab  Ag-Ab
Applying the Law of Mass Action
-association between CDR and monovalent Ag can
be expressed as:
Ag + Ab  Ag-Ab
K1= forward (Assoc) rate constant
K-1=reverse (dissoc) rate constant
Wherby K1/K-1=Ka
The Association/equilibrium constant
value of Ka depends on K1 for small haptens, K1 is
high for large proteins Ag’s K1 is lower
2. Avidity
-Avidity of antibody for an antigen
-the measure of the overall binding between the
antibody and a multivalent antigen
-the overall strength of binding between
multivalent Ag’s and Ab’s
-Avidity is influenced by both the valence of the
antibody and valence of Antigen
-Avidity is more than the sum of the individual
affinities
Factors Affecting Antigen-Antibody Interactions:
 Affinity-the higher the affinity of the antibody for
the antigen, the more stable will be the
interaction. Thus, the ease with which one can
detect the interaction is enhanced
 Antigen to antibody ratio: The ration
between the antigen and antibody influences
the detection of antigen-antibody complexes
because the size of the complexes formed is
related to the concentration of the antigen
and antibody.
 Physical form
of the
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 antigen- The physical form of the antigen
influences how one detects its reaction with
antibody. If the antigen is a particulate, one
generally looks for agglutination of the antigen by
the antibody. If the antigen is soluble one
generally looks for the precipitation of the antigen
after the production of large insoluble antigen-
antibody complexes.
3-distinct phases of Antigen-Antibody Reactions
1. Primary Phenomenon
-are non-covalent interactions occurring between
an antibody and a univalent antigen
2. Secondary phenomenon
-interactions that occur between antigen and
antibodies when the antigens are multivalent
3. Tertiary phenomenon
Methods Based on Primary Manifestations of Antigen-
Antibody Interactions:
 Immunofluorescence
-a technique whereby an antibody labelled with a
fluorescent molecule (fluorescein or rhodamine
or one of many other fluorescent dyes) is used to
detect the presence of an antigen in or on a cell
or tissue by the fluorescence emitted by the
bound antibody
 Direct immunofluorescent assay
-The antibody specific to antigen is directly tagged
with the
fluorochrome
- UV light is used
the measurement of radioactivity associated with
immune complexes.
-in any particular test, the label may be on either
antigen or the antibody
 Enzyme Immunoassay
Competitive RIA/ELISA for Ag detection
-by using known amounts of standard unlabelled
antigen, one can generate a standard curve
relating radioactivity (cpm) (Enzyme) bound
versus amount of antigen. From this standard
curve, one can determine the amount of antigen
in an unknown sample.
Noncompetetive
RIA/ELISA (Enzyme
linked
immunoabsorbent
assay) for Ag or Ab
-Noncompetitive RIA
and ELISAs are also
used for the
measurement of antigens and antibodies
-the bead is coated with the antigen and is used
for the detection of antibody in the unknown
sample. The amount of labelled second antibody
bound is related to the amount of antibody in the
unknown sample.

 Indirect Immunofluorescent assay
-the antibody specific is unlabelled and a second
anti-immunoglobulin antibody directed toward the
first antibody is tagged with fluorochrome.
-more sensitive than direct immunofluorescence
since there is amplification of signal.
- the bead is coated with antibody and is used to
measure an unknown antigen. The amount of
labelled second antibody.
- Measure any colored reaction

 Radioim
munoassay (RIA)
-are assays that
Methods Based on Secondary Manifestations of Antigen-
are based on
Antibody Interactions
 Precipitation
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-formation of immune complexes (precipitates)
due to antibodies interacting to soluble antigens
-when divalent Abs (IgG) and multivalent Abs
(IgM) can be cross-linked by multivalent antigens
-it occurs both in solutions and in semisolid media
such as agar gels
o Radial immunodiffusion (Mancini)
-antibody is incorporated into the agar gel as it is
poured and different dilutions of the antigen are
placed in holes punched into the agar. As the
antigen diffuses into the gel, it reacts with the
antibody and when the equivalence point is
reached a ring of precipitations is formed.
-the diameter of the ring is proportional to the log
of concentration of antigen since the amount of
antibody is constant.
o Immunoelectrophoresis
-a complex mixture of antigens is placed in a well punched
out of an agar gel and the antigens are electrophoresed so
that the antigen are separated according to their charge.
-after electrophoresis, a trough is cut in the gel and
antibodies are added
-As the antibodies diffuse into the agar, precipitin lines are
produced in the equivalence zone when an
antigen/antibody reaction occurs
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 Agglutination
o Direct Agglutination
-interaction of an antibody with a particulate
antigen leads to agglutination
-cross-linking of antigen particles by antibodies
leads to clumping or agglutination
-antigen and antibody are mixed in appropriate
proportions
a)Qualitative agglutination test
-antibody is mixed with the particulate antigen
and a positive test is indicated by the
agglutination of the particulate antigen
b) Quantitative agglutination test
-in this test, serial dilutions are made of a sample
to be tested for antibody and then a fixed number
of red blood cells or bacteria or other such
particulate antigen added.
-then the maximum dilution that gives
agglutination is determined.
*Titer-the maximum dilution that gives visible
agglutination
-results are reported as the reciprocal of the
maximal dilution that gives visible agglutination
*Prozone effect-occasionally, it is observed that when the
concentration of antibody is high (i.e. lower dilutions),
there is no agglutination and then, as the sample is
diluted, agglutination occurs.
-the lack of agglutination at high concentration of
antibodies
-Application of agglutination test:
1. Determination of blood types or antibodies to blood
group antigens
2. To assess bacterial infections
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 Passive Hemagglutination
-the agglutination test only works with particulate
antigen. However, it is possible to coat
erythrocytes with a souluble antigen (e.g. viral
antigen, a polysaccharide or a hapten) and use
the coated red blood cells in an agglutination test
for antibody to the soluble antigen.
 Transfusion reaction
o Indirect Coomb’s Test
-this test is done by incubating the red blood cells
with the serum sample, washing out any unbound
antibodies and then adding second-
antiimmunoglobulin reagent to cross link the
cells.

 Coomb’s Test
-looks for antibodies that may bind to your red
blood cells and cause premature red blood cell
destruction (hemolysis)
o Direct Coomb’s Test
-in order to detect the presence of non-
agglutination antibodies on red blood cells, one
simply adds a second antibody directed against
the immunoglobulin (antibody) coating the red
cells.
-this anti-immunoglobulin can now cross link the
red blood cells and result in agglutination
APPLICATIONS
-an abnormal (positive) indirect Coomb’s test
means you have antibodies that will act against
red blood cells your body view as foreign. They
may suggest:
 Detection of anti-rhesus factor (Rh)
antibodies
 Autoimmune or drug-induced haemolytic
anemia
 Incompatible blood match

APPLICATIONS
-An abnormal (positive) direct Coomb’s Test
means you have antibodies that act against your
red blood cells. This may be due to:
 Autoimmune haemolytic anemia
 Chronic Lymphocytic leukemia
 Drug-induced haemolytic anemia
 Erythroblastosis fetalis
 Infectious mononucleosis
 Mycoplasma infection
 Syphilis
 Systemi lupus erythematosus
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 Complement -for example, if 1 mL of serum is added to 10 mL of H2O,
Fixation this means 1mL + 10mL yields a total volume of 11mL
-an -This means a 1/11 dilution was made.
immunological
medical test that
can be used to Serial dilution
detect the -this term is frequently used and refer to a “multiple”
presence of dilution problem.
either specific -In other words, an initial dilution is made and then this
antibody or dilution is used to make a second dilution and so on.
specific antigen
in a patient’s Preparation of Dilutions
serum 1. The first rule is performing dilution is careful
reading of procedural instructions
2. If several dilutions are made in succession of one
another, the final dilution can be calculated by
simply multiplying each dilution factor involved.
-Methodology: *Always reduced the final answer to a fraction
 Ag mixed with test serum to be assayed with the lowest possible denominator
for Ab
 -Standard amount of complement is Serodiagnosis of Syphilis
added
 Erythrocyte coated with Abs is added History:
 Amount of erythrocyte lysis is determined
 The “pre-columbian theory” states that syphilis
was present in Europe prior to the voyage of
Serial Dilutions
Columbus
-many of the laboratory procedure involve the use of  Some believed that the disease first appeared in
dilutions Central Africa and was carried by travellers and
-it is important to understand the concept of dilutions, traders
since they are a hand tool used throughout all areas of the  The “Columbian theory” states that syphilis was
clinical laboratory endemic in Haiti (Hispaniola) and was carried to
-most common serial dilutions performed: Europe by the crew of Columbus
 Two-fold dilution  In 1495 the disease was first mentioned in the
 Five-Fold dilution medical literature
 Ten-Fold Dilution  In 1497 mercury was advocated as treatment
 In 1905 the causative spirochete was discovered
Dilution and named Spirochaeta pallida. Later, the name
-are expressed as the ratio of the quantity of a desired changed to Treponema pallidum
solute (serum, urine, chemical solution, etc.) contained in  In 1906 the Wasserman test was described
a solvent (diluent)  Several treatments were advocated including
Ex: 1: 10 dilution of serum was made by adding one part salvarsan 606 (known as asphenamine), neo-
serum to nine parts diluent to make a total of ten parts arsphenamine, and bismuth
 In 1943 penicillin was found to be a miraculous
Added to cure
-it is not the same as “diluted to”  The prevalence of Syphilis has fluctuated over the
-it refer to the volume of the solute added to a specified years. About 30-40 percent of cases were found in
volume of solvent homosexuals or bisexuals males.

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Morphology of Treponema pallidum
-a member of the order Spirachaetales, the family
Treponematoceae, and the genus treponema
-The family contains three genera: Borelia, Treponema,
Leptospira
-The genus Treponema contains four principal species of
pathogenic organisms, T. pallidum, T. pertenue, T.
carateum, and T. cuniculi
-T. pallidum subspecies pallidum, the etiologic agent of
venereal syphilis, is a thin (0.2 micrometes) spirochete; 6-
20 micrometer in length with 10-13 coils
-the organism is believed to multiple by transverse fission
-In aquaeous media, young treponemes spin vigorously
aroung their long axis in an apparently useless type of
motion. In viscous media, they propel themselves/
-The spirochete has corkscrew motility with an undulating
central flexion
Metabolism of Treponema pallidum
-Outside the host, T. palidum is susceptible to a variety of
physical and chemical agents that rapidly bring about its
destruction. It is readily killed by heat, cold, desiccation,
disinfectants, and osmotic changes
-the treponemes have a complex nutritional
requirements. They require use of glucose as a primary
source of energy, as well as other AA, purines and
pyramidines. Some strains also need bicarbonate, co-
enzyme and fatty acids
-Under anaerobic conditions in various media, the
organism can remain viable and motile for up to 15 days
at 35 degrees C
-T.pallidum has not been recovered in blood, serum or
plasma stored at 4 degrees for more than 48 hrs.
-Treponemes can be kept viable when frozen at -70
degrees C for years
-But humoral and cell-mediated immune response are
involved in the interaction between T. pallidum and the
human host. Antibodies are produced, there is an
inflammatory response, immune complexes are formed,
and the lesion is walled off. The disease then enter the
latency stage
Antigens
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-there are two types:
The Wasserman Antigen
-the antigen used in wasserman test is cardiolipin
-Free cardiolipin is a hapten and is bound to the
microbial cell in order to be antigenic
-the microbial cell is foreign carrier and the bound
cardiolipin is the immunogenic determinant
The Treponamal Antigens
-in the treponemes, two classes of antigens have
been recognized: those restricted to one or more
species and those shared by many different
spirochetes.
-The Reiter treponeme (reputed to be a cultivable,
non-virulent variant of T. pallidum) can be used as
antigen
Stages of Syphilis (correlation with test result)
1. Syphilis is ordinarily transmitted by Sexual Contact
2. In males, T. pallidum organisms are present in
lesions on the penis or discharged from deeper
genitourinary sites along with the seminal fluid
3. In females, the lesions are commonly located in
the perianal region or on the labia, vaginal wall, or
cervix.
4. The primary infection is extragenital (usually in or
about the mouth) in 10% of cases
Primary Stage/Early Stage
1. T. pallidum enters the skin through small breaks,
but can pass through intact mucous membranes
where it is carried by the blood stream to every
organ of the body
2. A chancre (painless ulcer) develops at the site of
entrance within 10-60 days
3. The primary chancre persists for 1-5 weeks, then
heals
4. Serum tests for Syphilis is usually positive
between the first and third week after the
apparent chancre
Secondary Stage
1. This usually occurs 6-8 weeks after the chancre
appears-in one third of cases, it occurs before the
chancre disappears
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 2. Symptoms are generalized rash (often involving
mucous membranes) and lesions in eyes, joints, or
central nervous system
3. Secondary lesions contains large numbers of
spirochetes. When located on exposed surfaces,
they are highly contagious.
4. The lesions subside spontaneously after 2-6
weeks.
5. Serologic tests are invariably positive
6. Sometimes (rarely) primary and secondary stages
go unnoticed
The Late Latent Stage
1. This occurs after about second year of infection
2. The disease is contagious during this stage
3. There are no clinical sign or symptoms
4. Serologic tests are positive
5. This stage may last for years or even for the rest of
persons’s life
6. After 4 years the disease is rarely communicable,
except between mother and fetus
Tertiary Stage
1. Lesions are usually seen from 3-10 years after
primary stage (or later)
2. Lesions (gummata) are usually located on the
skin, mucous membranes, subcutaneous and
submucous tissues, bones, joints, muscles and
ligaments
3. Lesions are serious when present in the nervous
system, the cardiovascular system, and the eyes
4. In about one-fourth of untreated cases, this stage
is asymptomatic
5. Serologic tests are usually positive-occasionally
negative
Antibodies in Syphilis
Development
I. Infected individuals produce specific and non-
specific antibodies
II. Specific antibodies are directed against T.
pallidum
III. Non-specific antibodies are directed against the
protein antigen group common to pathogenic
spirochetes
IV. Specific antibodies in early or untreated early
latent Syphilis are predominantly of IgM
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V. Levels of IgG remains constant during the
secondary and early latent stage-basic IgG
decreases
VI. Nonspecific IgA antibodies increase significantly
during the course of untreated syphilis.
Serologic Test for Syphilis
-Serologic Tests for Syphilis on the detection of one or
more of the antibodies produced
-There is no single ideal test
-There two known types of host antibodies:
A. Reagin Antibodies (non-treponemal), which react
with lipid antigens
B. Treponemal antibodies-which react with T.
pallidum and closely related stains
-Reagin test includes VDLR slide test, rapid plasma regain
test (RPR), Plasmacrit test (PCT), unheated serum regain
test, RPR circle card test, and the automated regain test
-Treponemal test include T. pallidum immobilization test
(TPI), Treponema pallidum complement fixation (TPCF),
Reiter protein complement fixation test (RPCF),
Fluorescent treponemal antibody test, fluorescent
treponemal antibody absorption test (FTA-ABS), T.
pallidum hemagglutination test (TPHA), Direct Fluorescent
antibody test (DFA)
C-reactive Protein
-C-reactive protein (CRP) is a nonspecific trace constituent
of serum
-CRP appears in the sera of individuals in response to a
variety of inflammatory conditions and tissue necrosis and
disappears when the inflammation condition has subsided
-CRP is consistently found in bacterial infection, active
rheumatic fever, and many malignant diseases
-CRP is commonly found in active rheumatoid arthritis,
viral infection, and tuberculosis
-CRP has been detected in patients following surgical
operations and blood transfusions.
-Bullous fluid aspirated from patients with thermal burns,
pemphigus vulgaris, and other bulbous lesions has been
found to contain CRP
-CRP appears after the onset of disease (14-26 hrs)
-it may increase in concentration by as much as 1,000
times its normal amount and is therefore, a useful clinical
indicator of disease states.
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-Testing for CRP is complicated by nonspecificity and by a. Agglutination Tests-using latex particles coated
lack of correlation between positivity of the test and the with antibodies to human CRP
severity of the disease. –Also known as latex-fixation test
-The biologic significance of CRP is not clear –Claimed to be most sensitive
-Properties: b. Complement Fixation tests-not generally used in
 Human CRP is a homogenous molecule (MW: the routine laboratory
120,000) c. Fluorescent antibody tests-primarily a research
 It has a sedimentation coefficient of 6.5 test, useful in studying binding of CRP to
 Its electrophoretic mobility is in the gamma lymphocyte and their subpopulations and used in
region localizing CRP in tissues
 -it consists of five probably identical non- d. Precipitaion-either in fluid hase (by capillary or a
covalently bound subunits of approximately 21, tube methor or solid gel phase (by the
500 to 23, 500 d each, linked in the form of a Ouctherclony, Oudin, or Mancini Method)
cyclic pentamer e. Radioimmunoassay
 It is made up of 100 percent peptide
 It has an amino acid composition similar to that of
IgG
 It shares with immunoglobulin the ability to
initiate precipitation, agglutination, opsonisation,
capsular swelling, and complement activation
 It binds with and inhibits certain T-lymphocyte
functions
 It inhibits the aggregation of platelets induced by
aggregated human gamma globulin and thrombin
 It differs from immunoglobulin in antigenicity,
tertiary structure, homogeneity, required stimuli
for formation, and release and binding
specificities, and that it is produced entirely by
hepatocytes or liver parenchymal cells
 CRP is thermolabile-it is destroyed by heating to
70 degrees C for 30 minutes
 CRP does not cross human placenta
 CRP elevation above normal (0.5mg/dL) indicates
tissue damage or inflammation or both.
Sequential monitoring aids in assessing disease
activity and the guiding of therapy

Current and potential uses of CRP Determination

1. CRP is early, reliable indicator of clinical disease
compared to other plasma proteins
2. CRP test can be useful as a means of evaluating
the postoperative clinical course of a patient
3. CRP tests are useful in evaluating the clinical
course of bacterial and viral infections, rheumatic
diseases, myocardial infarction, burn injuries, and
renal transplantation

Laboratory Test for CRP

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