Antigen – Antibody Reactions or Serological Reactions

Antigen-Antibody interactions Characterized as: Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate) Do not lead to irreversible alteration of Ag or Ab This exact and specific interaction has led to many immunological assays that are used to: detect Ag or Ab diagnose disease measure magnitude of humoral IR identify molecules of biological and medical interest

Introduction Ag – Ab reactions are one of the most specific noncovalent biochemical reactions known The forces that hold the reactants together are: - van der Waals forces - Electrostatic forces - Hydrophobic forces They can be represented by the simple formula: Ag + Ab ↔ AgAb The reaction is driven to the right but it is reversible

Strength of Reaction The strength of the reaction (how far it is driven to the right) is referred to as affinity Antibody affinity - A quantitative measure of binding strength - Combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag - Affinity varies broadly among immunoglobulins

Strength of Reaction Antibody avidity - Avidity is often used to describe the collective affinity of multiple binding sites on an antibody molecule - True strength of the Ab -Ag interaction within biological systems - The interaction at one site will increase the possibility of reaction at a second site - High avidity can compensate for low affinity (secreted pentameric IgM has a higher avidity than IgG )

CROSS REACTIVITY Antibody elicited by one Ag can cross-react with a related Ag. Occurs if two different Ags share identical or very similar epitope 1- Vaccinia virus and smallpox virus 2- Rabies & JE vaccine 3- Streptococcus pyogenes infection: heart & Kidney damage following infection 4- Original antigenic sin. 5- Bacterial Ag and sugars on RBC

STAGES OF Ag - Ab REACTIONS Primary reactions Vs secondary reactions: Small Ag - Ab complexes Vs large complexes (The Lattice hypothesis) Development of macroscopic manifestations reactions (e.g. immunoprecipitation) Ag – Ab reactions involving IgM are confined to the blood stream, while those of lower molecular weight (IgG and IgE) can leave the vasculature and enter tissues Time required is hours to days for precipitin formation leading to irreversible immunoprecipitates

LATTICE THEORY Lattice formation (visible Ag - Ab aggregates) occurs when: Ag is multivalent (contains more than 2 identical epitopes) Cross-linking of Ags by specific Abs (2 or more antigen-binding sites) Molar ratios of epitopes and antigen-binding sites are optimal (zone of equivalence)

Zone of equivalence

LATTICE THEORY Zones of lattice formation Far Ag excess (no ppt. formed; free Ag in supernatant) -- “postzone” Ag excess (sub-optimal ppt.; free Ag in spnt.) Zone of equivalence (maximum ppt.; no Ag or Ab in spnt.) Ab excess (sub-optimal ppt; Ab in spnt.) Far Ab excess (no ppt; Ab in spnt.) -- “prozone”

ZONES OF PRECIPITIN FORMATION Precipitin Curve

METHODS THAT DETECT Ag- Ab REACTIONS Primary Reactions: - Immunofluorescence (IF) - Radioimmunoassay (RIA) - Enzyme immunoassay (EIA) - Immunonephelometry (measures picogram to nanogram quantities of analyte) Secondary Reactions - Agglutination Techniques - Precipitation Techniques ± Electrophoresis

Precipitation Precipitation can take place in capillary tubes, test tubes, and in gel

Precipitation in gel - Double diffusion - Single (radial) diffusion - Combination of diffusion in gel and electrophoresis

SINGLE VS. DOUBLE DIFFUSION Single diffusion Supporting medium (gel) contains one reactant at a uniform concentration Only the unknowns move through the medium Double diffusion Gel is inert (contains no reactants) Both Ag and Ab travel through the medium

The region of equivalence

RADIAL IMMUNODIFFUSION Ab uniformly distributed in gel; Ag diffuses outward from a well (single diffusion) Ag- Ab complexes form as concentric rings around the well at zone of equivalence At a set time, ring diameters are measured [Ag] is directly proportional to the ring d2 Unknown value is determined by comparing to a 3-standard curve

RADIAL IMMUNODIFFUSION Samples Standards Precipitin Rings A B C a b c Standard Curve

RADIAL IMMUNODIFFUSION Fahey method (kinetic) Read at 18 hours Plot [std] vs. ring diameter on semi-log paper Mancini method (endpoint) Read at 48 or 72 hours Plot [std] vs. ring diameter squared on graph paper Results reliable only if the ring size is within the range of the standards; if greater than highest std, dilute and repeat test Used to measure IgM, IgG, C4,C3,transferrin, CRP, others

OUCHTERLONY DOUBLE DIFFUSION Ag & Ab placed in wells cut into an agarose gel (both reactants diffuse) Precipitin line (or arc) indicates Ab has specificity for Ag Position of precipitin between wells depends on MW and concentration of reactants 3 possible patterns of reaction: identity, non-identity, partial identity

OUCHTERLONY DOUBLE DIFFUSION Ouchterlony Plates Precipitin Patterns

ELECTROIMMUNOASSAY (ROCKET) Electrophoresis hastens movement of Ag (placed in wells) through Ab -imbedded gel (single diffusion) Selected pH (8.6) keeps Abs at their isoelectric point; they will not move Rocket-shaped precipitin bands will form at zone of equivalence (changes as reactants move) [Ag] proportional to length of rocket Unknowns compared to standards

ELECTROIMMUNOASSAY (ROCKET)

ELECTROIMMUNOASSAY (ROCKET) May be used to quantitate plasma proteins such as coagulation factors, alpha- fetoprotein, C3, C4, CRP, haptoglobin Compared with RID: faster similar sensitivity requires electrophoretic equipment and more technological finesse Largely replaced by immunonephelometry

IMMUNONEPHELOMETRY Ag + Ab  AgAb  microscopic Ag - Ab complexes Microcomplexes cause light moving through the suspending solution to scatter Nephelometer detects light scattered at a 90o angle Amount of light scattered at 90o is proportional to Ag - Ab complexes formed Sensitive and quantitative technique used for measurement of many serum proteins

IMMUNOELECTROPHORESIS (IEP) Electrophoresis and double diffusion 2 stages Proteins separated by electrophoresis Antiserum placed in trough parallel to separated proteins; all reactants diffuse in all directions Precipitin forms at zones of equivalence Trough may be filled with simple or complex antisera yielding simple to complex patterns

Immunoelectrophoresis of normal human serum. 33 Immunoelectrophoresis of normal human serum.

IMMUNOELECTROPHORESIS (IEP) Qualitative to semi-quantitative Serum, urine, or CSF may be analyzed Complex patterns may be difficult to interpret Useful to detect: missing proteins abnormal proteins normal proteins in abnormal concentrations Used to evaluate conditions such as multiple myeloma Largely replaced by immunofixation

IMMUNOFIXATION ELECTROPHORESIS (IFE) Proteins that were separated by electrophoresis are exposed to Ab directly, instead of through diffusion Steps: Electrophoresis of protein mixture in gel (use serum or urine samples) Paper strips imbedded with specific Ab are “blotted” onto gel; Ags transfer to paper and bind to Abs Strips washed (unbound material washes away) Strips stained to reveal precipitin bands

IFE

IMMUNOFIXATION ELECTROPHORESIS (IFE) Used to detect the presence of Igs in conditions like multiple myeloma Fairly sensitive - Ab is highly specific, electrophoresis leaves Ag isolated and accessible Faster and easier to interpret than IEP Only 1 Ab may be used per strip

WESTERN BLOTTING Similar to IFE but the unknown is Ab rather than Ag Steps: Separation of complex antigenic material (eg., viral proteins) by electrophoresis Separated components transferred from gel to nitrocellulose paper by “blotting” Unknown (or control) sera (which may have Abs) incubated with paper strips; Ag - Ab complexes ppt. at site of transfer Strips washed; staining reveals complexes

WESTERN BLOTTING

The Western blot procedure. 42

FLOCCULATION Immunoprecipitation (or agglutination) of insoluble particles Characterized by very sharp pro- and postzones No precipitin formed in zones of Ab or Ag excess, only in zone of equivalence Clinically important examples, VDRL and RPR tests (screening tests for syphilis)

FLOCCULATION VS. IMMUNOPRECIPITATION

VDRL (Venereal Disease Research Lab.) test Flocculation Tests VDRL (Venereal Disease Research Lab.) test RPR (Rapid Plasma Reagin) test

Agglutination Titer Zeta potential Types of Agglutination - Direct agglutination or hemagglutination - Indirect (passive) agglutination or hemagglutination - Agglutination or hemagglutination inhibition The Coombs test - Direct - Indirect

Agglutination Reactions

Agglutination Qualitative slide agglutination - identification of bacteria with antisera directed against O, H, K antigens

Agglutination Latex agglutination Coagglutination

Agglutination Tube agglutination tests: - Gruber-Widal: typhoid fever (S. typhi) - Weil-Felix: typhus (Rickettsia) - Wright: brucellosis Identify and titrate antibodies in the patient’s serum. Titre: is defined as the reciprocal of the highest dilution of serum showing agglutination.

1:200 1:400 1:100 Titer

Agglutination inhibition

Hemagglutination Inhibition Test To Detect Antibodies (Rubella) - Serum (Ab)+ HA +RBCs= No Hemagglutination = Positive Test - Serum (No Ab)+ HA + RBCs =Hemagglutination =Negative Test To Detect Antigen (HBsAg) - Serum (HBsAg) +Anti HBsAG + HBsAg coated RBCs = No Hemagglutination = Positive Test - Serum (No HBsAg)+ Anti HBsAG + HBsAg coated RBCs = Hemagglutination =Negative Test

Use of Labels in Ag – Ab Reactions Immunoassays - Radioimmunoassay (RIA) - Enzyme Immunoassys (EIA) Immunofluorescence (IF) - Direct IF - Indirect IF Flow cytometry and Cell Sorting (FACS)

Immunologic Tests 4) Radioimmunoassay (RIA)– a very sensitive test; used for measuring hormones, serum proteins, drugs, etc. at low concentrations (≤ 0.001ug/ml) measures “competitive binding” of radiolabelled Ag + unlabelled (test) Ag to high affinity Ab

ELISA

ELISA tests Depend on enzyme conjugated to 2 Ab reacting with a specific substrate to produce a color reaction. Variations of ELISA’s: Allows for qualitative or quantitative testing. Each one can be used for qualitative detection of Ag or Ab Also, a standard curve based on known concentrations of Ag/Ab can be prepared and an unknown concentration can be determined Indirect ELISA Sandwich ELISA Competitive ELISA

Direct and indirect Immunofluorescence

Immunoprecipitation Provides a quick and sensitive test for finding proteins/Ag’s especially in low concentrations Binds Ab to synthetic bead support  centrifuged Or 2° Ab with bead or magnetic bead and collect by magnetism

Distribution of selected markers on some leukemia cell types → Immunophenotyping using “flow cytometry & mAb”

Sensitivity of various immunoassays