IS LAB ACTIVITY 3A & 3B

Pathogens and Antigens

 Bacteria, viruses, fungi, parasites and infectious proteins are some pathogens capable of causing infectious diseases.
 Antigens usually associated with these pathogens
 Difference between pathogens from antigens and vice cersa?
o Antigen
 Specific reactivity – ability to react specifically with the antibodies or cells it provoked
 Immunogenicity – ability to provoke an immune response by stimulating production of antibodies, proliferation of specific
T cells, or both
o Associated terms:
 Immunogen – capable of inducing an immune response
 Hapten – no immunogenicity but has reactivity
 Simple or nonprecipitating – combine with antibody; cannot produce precipitates
 Complex or precipitating – combine with the antibody and produce precipitates
 Carrier/schlepper molecules – larger molecules attached to haptens which confer new antigenic specificities

COMPLETE ANTIGEN HAPTEN/INCOMPLETE ANTIGEN

- Capable of stimulating antibody synthesis in the host and can - Can’t stimulat an immune response independently
also react with homologous antibodies - Can react specifically with homologous antibodies
- Bacterial cells and protein

Antibody or Immunoglobulin (Ig)

 The substance produced in response to antigenic stimulation
 Capable of specific interaction with provoking immunogen
 General Function:
o Combine with antigens on cellular surfaces and cause the destruction of these cells either
 Extravascularly - outside of the blood vessels within the mononuclear-phagocyte system
 Intravascularly – within the blood vessels thru action of the complement
o Facilitate phagocytosis and kill microbes
o Neutralize toxic substances
Structure of the Basic Immunoglobulin Unit

Note that:

 VH = variable region heavy chain  CH = constant region heavy chain

 VL = variable region light chain  CL = constant region light chain
Basic Structure  Four chain polypeptide unit
o Consist of two heavy chains and two light chains held together by disulfide bonds
 2 heavy chains – __ amino acids
 2 light chains - ___ amino acids

Heavy chains  Always of the same type (immunoglobulin molecule

 Determine the immunoglobulin class:
o Alpha
o Gamma
o Delta
o Epsilon
o Mu
Light chains  Kappa and lamba
 Both kappa and lamba are found in all classes of immunoglobulins
 Only one type is present in a given molecule
Disulfide bonds  Holds each light chain to a heavy chain
 Link the mid-region of the two heavy chains
Fab fragment  Fragment antigen-binding
 Consist of 1 light chain and ½ of a heavy chain
 Intact immunoglobulin has 2 fab fragments, each representing 1 antigen-binding site.
Fc fragment  Fragment crystalline
 Carboxy-terminal end halves of the two heavy chains
 Has no antigen-binding ability
Constant region  Carboxy terminal end of the immunoglobulin molecule  amino acid sequence is the same for all chains
of that type
 Responsible for the type and antigen-antibody reaction that occurs
 Constant region of heavy chain differs from one antibody class to the other
Variable region  Amino-terminal end of the immunoglobulin molecule  amino acid sequence varies
 Responsible for the specificity of a particular immunoglobulin
 Different for each antibody molecule
Hypervariable region  Regions within variable region
 Form the antigen-binding site
 Through changes in the hypervariable region, an immense diversity of antigen-binding sites can be
created
 Valence: number of binding sites
Hinge region  Flexible portion of the heavy chain
 Located between first and second constant regions
 Allows the molecule to bend to let 2 antigen-binding sites operate independently
Joining region  Glycoprotein
 Serves to link immunoglobulin monomers together
 J chain is found in: ______

ISOTYPE
 Same heavy chain for each class
 H chains – unique to each
immunoglobulin class
Antibody fragmentation
ANTIBODY VARIATION
ALLOTYPE IDIOTYPE
 Variation in constant regions  Variations in variable regions
 Genetic variations in the constant  Variations in variable regions that
regions give individual antibody molecules
specificity
COMPLEMENT SYSTEM

 A defensive system consisting of over 30 proteins produced by the

liver and found in circulating blood serum
 Most are inactive enzyme precursors that are converted to active
enzymes in a precise order
 It works like a cascade system  when one reaction triggers
another reaction which triggers others and so on; these types of
systems can grow exponentially very fast

MAJOR FUNCTIONS

1. Opsonization
 Following activation, opsonization occurs as complement
components coat pathogenic organisms or immune
complexes
 Facilitating the process of phagocytosis
2. Inflammation
 Activation of the complement system results in the
induction of histamine release from mast cells and
basophils, and stimulation of inflammatory response
3. Cytotoxic
 In the final stage of the complement cascade membrane
attack of target cells (e.g. bacteria and tumor cells) occur,
leading to cell death

PATHWAY OF COMPLEMENT ACTIVATION

CLASSICAL PATHWAY ALTERNATE PATHWAY MANNOSE-BINDING LECTIN PATHWAY

 Involves more recently evolved  Provides nonspecific innate immunity  Involved attachement of mannose-
mechanism of specific adaptive  Considered a primitive defense binding lectin to mannose residues on
immunity mechanism glycoproteins or carbohydrates on the
 Initiated by the presence of antigen- o Bypass mechanism that does surface of microorganisms
antibody complex (immune complex) not require C1, C4, and C2
 Typically requires suitable bound to an interaction PROTEINS:
antigen, complement components 1, 4, 2  Antigen-antibody complex is not necessary MBL Binds to mannose
and 3 and calcium (Ca++) and for it to take place MASP-1 Helps cleave C4 and C2
Magnesium (Mg++) cations  Begins with the activation of C3 and MASP-2 Cleaves C4 and C2
 Requires interaction of all 9 major requires factors B and D, and magnesium
complement components. cation (Mg++) MBL = Mannose Binding Lectin
 All present in normal serum MASP-1 = MBL-associated Serine Protease-1
PROTEINS: MASP-2 = MBL-associated Serine Protease-2
C1q Binds to Fc of IgM and IgG PROTEINS:
C1r Activates C1s Factor B Binds to C3b to form C3
C1s Cleaves C4 and C2 convertase
C4 Part of C3 convertase Factor D Cleaves factor B
C2 Binds to C4b Properdin Stabilizes C3 convertase
C3 Key intermediate in all pathways
C5 Initiates membrane attack complex
(MAC)
C6 Binds C5b in MAC
C7 Binds C5bC6 in MAC
C8 Starts pore formation on membrane
C9 Polymerizes to cause cell

C1: Recognition Unit
CLASSICAL PATHWAY
 Trimolecular complex (C1q, C1r, and C1s) held together by Ca+2 ions
 C1q is the largest and consists of 6 globes held on slender shafts that fuse common base
 6 globe act as recognition units that bind to the Fc region of IgM and IgG
 For C1q to initiate the cascade, it must attach to 2 Fc fragments from IgG or IgM

C4: First activation unit
Activation Sequence:
o C1q attaches to the immunoglobulin and initiates complement activation  C1q binding initiate C1r
 C1 r cleaves C1s
 A beta globulin originates from a proC4 synthesized by the macrophage
 Consist of 3 peptide chains (C4-a, C4-b, and C4, Y) joined by disulfide bonds
 C4 is cleaved into C4a and C4b by C1s

Activation Sequence:
o C1s mediates cleavage of C4 into C4a and C4b  C4b is bound to cell membrane while C4a is
released into the fluid phase
C2: Second activation unit  C2 is cleaved into C2a and C2b by C1s in the presence of C4b

C3: Third activation unit
Activation Sequence:
o C1s in the presence of C4b (C14b) cleaves C2 units into C2a and C2b
o C2a is bound to the cell-bound C4b while C2b is released in the fluid phase
o C4b2a complex is now attached on the surface of the cell membrane
 Most abundant complement component in the serum
 Cleaved by C3 convertase into C3a and C3b

Activation Sequence
o C3 is cleaved by C3 convertase into C3a and C3b
o C3a remains unbound
o C3b is bound to the cell-bound C4b2a
o C4b2a3b complex is now attached on the surface of the cell membrane
C5: First membrane attack  C5 is cleaved by C5 convertase into C5a and C5b
unit
Activation Sequence:
o C5 is cleaved by C5 convertase into a smaller C5a and a larger C5b
o C5a is released into the surrounding fluid medium
o C5b is the first component of the membrane attack complex that is bound on the surface of the cell
membrane that serves as the receptor of C6 and C7
C6: Second membrane Activation Sequnece:
attack unit o C6 binds to cell-bound C5b forming a stable C6b6 complex
C7: Third membrane Activation Sequnece:
attack unit o C7 binds to cell-bound C5b6 forming the stable C5b6 forming the stable C5b6 that is bound to the
target cell membrane
C8: Final membrane attack Activation Sequnece:
unit o C8 binds to C5b67 complex and leakage of membrane begins
o Cell lysis can occur by the C5b678 complex in the absence of C9
C9: Final membrane attack Activation Sequnece:
unit o C9 binds to cell-bound C5b678 complex accelerates cytolysis by producing circular lesions in the
membrane
o C5b6789 complex induces the formation of hollow cylinders (tubules) in the bilipid layer of the cell
membrane allowing exit of electrolytes and water out of the cell
ALTERNATE PATHWAY
1. The initial recognition necessary for the alternative pathway is the presence of C3, specifically C3b that is probably continuously generated
in small amounts in the circulation
2. C3 activation, where C3b that exists in trace amounts in normal serum becomes membrane-bound on the surface of target cells, happen
through any of the following:
 Non-immunogenic
o Lipopolysaccharide (LPS)
o Endotoxin from the cell walls of gram-negative bacteria
o Cell walls of some bacteria
o Cell walls of yeasts (zymosan)
o Cobra venom factor (CVF)
  Immunogenic
o IgA
o Other antibodies
o Óther antibody types can fix the alternate complement pathway?
3. The membrane-bound C3b interacts with Factor B (C3 proactivator) to form C3bB, which is a magnesium ion-dependent complex
4. C3bB is cleaved by Factor D (C3 proactivator convertase) into 2 fragments, Ba and Bb
5. Ba is released, and Bb is bound to C3b forming the C3bBb complex: amplification C3 convertase
6. When stabilized by Properdin (Facotr P), the C3bBb complex becomes the C3 convertase that cleaves C3 into C3a and C3b
7. As more C3b is generated, the complex expands (C3bnBb) and becomes a C5 convertase (e.g., C3bBb3b)
8. C5 convertase cleaves C5 into C5a and C5b initiating the membrane attack pathway
9. Membrane attack complex (C5b6789)
MANNOSE-LECTIN BINDING PATHWAY
1. Macrophages that digest microbes release chemicals that cause liver to produce lectins
 Lectins are mannose-binding lectin or MBL which proteins that bind to carbohydrates such as mannose on the surface of
the microbe
 The liver then produces MBL in acute-phase inflammatory reactions
 MBL acts like C1q of the classical pathway but bi7nds to mannose on many bacterial cells
2. Once MBL binds to the target cell, 2 serine proteases (MASP-1 and MASP-2) bind to the bacterial surface.
 MASP-1 & MASP-2 act like C1r and C1s of the classical pathway, respectively
3. Cleaving of C4 and C2 to form C3 convertase
4. Cleaving of C3 forming C5 convertase
5. Cleaving of C5 initiating the formation of the membrane attack complex (C5b6789)

CATEGORIES OF SEROLOGIC TESTS:

o To detect unknown antibody in serum (specimen) using known commercial antigen (reagent)
o Tests to detect unknown antigen (specimen) using a known commercial antiserum (reagent)
ANTIGEN-ANTIBODY REACTIONS:
o Forces that participate in antibody-antigen interaction
o Electrostatic forces (ionic bonds)
o Van der Waals Forces – London dispersion forces
o Chemical bonds
 Hydrogen bonding
 Hydrophobic bonding
 Bond not involved in antigen-antibody reactions:
Note: Factors affecting forces of attraction
o Physiologic pH
o Salt concentration
o Temperature
AFFINITY AVIDITY
o Binding strength between an epitope and its o Strength of the bond between antigen and antibody
corresponding complementary site of the antibody o Overall binding strength
o Determined by the three-dimensional fit and molecular o The sum of affinities for all the antigen-binding sites in
attractions between one antigenic determinant and one one antibody molecule
antibody-binding site, as presented by the image on the
right

SPECIFICITY CROSS-REACTIVITY
o Antibody’s highest affinity for a particular antigen o Occurs when the antibody combines with an antigen that
is structurally similar to the immunogen that is
stimulated the antibody production or the antigen the
antibody has the highest affinity for (i.e. heterophile
antibodies)

ZONE OF EQUIVALENCE ZONAL PHENOMENA

The titer or level of antibody in serum, can be measured by using known antigens
The area where antigens and antibodies are approximately equal o Prozone phenomenon
an visualization of the reaction is optimized by either o Concentration of the antibody exceeds that of
agglutination or precipitation the antigen in solution
o Response:
o Postzone phenomenon
o Concentration of the antigen exceeds that of
the antibody solution
o Response:
 TYPES OF IMMUNOLOGIC REACTION/IMMUNE PHENOMENA
Immune Phenomena Explanation Application
Primary Phenomena o Specific recognition o Not easily detectable
o Combination of Ag and Ab o Test require either a purified Ag or
Ab
o Can be measured indirectly by
radioimmunoassay (RIA), enzyme
immunoassay (EIA),
immunofluorescence
Secondary Phenomena o Conformations of the amino acid o Measured more readily
chains resulting from interchain
hydrogen bonding
o Precipitation, agglutination,
complement fixation
Tertiary Phenomena o In vivo reactions o Some are useful diagnostically
o Inflammation
o Phagocytosis
o Deposition of immune
complexes
o Immune adherence
o Chemotaxis
o Involve the folding of polypeptide
chains through hydrophobic and
hydrogen bonds
Quaternary o Association of polypeptide subunits
to form one protein

o What is an example of quaternary immune phenomenon and its significance in the diagnosis of immunologic conditions?

o What are the effects of pH, temperature, and salt concentration in the in vitro reactivity of the different types of antibodies?