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

IntroductionAg – 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 ReactionAntibody 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 REACTIVITYAntibody 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 Latticehypothesis)

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 THEORYZones 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

PrecipitationPrecipitation 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

Standard Curve

Precipitin RingsA B C a b c

Standards Samples

RADIAL IMMUNODIFFUSIONFahey 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

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

42The Western blot procedure.

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

Flocculation Tests

- VDRL (Venereal Disease Research Lab.) test

- RPR (Rapid Plasma Reagin) test

AgglutinationTiter

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.

1:100 1:200 1:400

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

ImmunoprecipitationProvides 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