Antigen – Antibody ReactionsAntigen – Antibody Reactions

or or Serological ReactionsSerological Reactions

Antigen-Antibody interactionsAntigen-Antibody interactions

Characterized asCharacterized as::

Non-covalent interaction (similar to “lock and Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate)key” fit of enzyme-substrate)

Do not lead to irreversible alteration of Ag or AbDo not lead to irreversible alteration of Ag or Ab

This exact and specific interaction has led to This exact and specific interaction has led to many immunological assays that are used to:many immunological assays that are used to:

detect detect AgAg or or AbAb

diagnose diagnose diseasedisease

measure magnitude of measure magnitude of humoral IRhumoral IR

identify molecules of identify molecules of biologicalbiological and and medicalmedical interest interest

IntroductionIntroductionAg – Ab reactions are one of the most specific Ag – Ab reactions are one of the most specific noncovalent biochemical reactions knownnoncovalent biochemical reactions known

The forces that hold the reactants together are: The forces that hold the reactants together are: - van der Waals forces- van der Waals forces - Electrostatic forces- Electrostatic forces - Hydrophobic forces- Hydrophobic forces

They can be represented by the simple formula: They can be represented by the simple formula: Ag + Ab ↔ AgAbAg + Ab ↔ AgAb

The reaction is driven to the right but it is reversibleThe reaction is driven to the right but it is reversible

-

Strength of ReactionStrength of Reaction

The strength of the reaction (how far it is driven The strength of the reaction (how far it is driven to the right) is referred to as affinityto 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 immunoglobulinsAffinity varies broadly among immunoglobulins

Strength of ReactionStrength of ReactionAntibody avidity

- Avidity is often used to describe the collective affinity Avidity is often used to describe the collective affinity of multiple binding sites on an antibody moleculeof 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 STAGES OF Ag - AbAg - Ab REACTIONSREACTIONS

Primary reactions Vs secondary reactions: Small Ag - Primary reactions Vs secondary reactions: Small Ag - Ab complexes Vs large complexes (The Ab complexes Vs large complexes (The LatticeLattice hypothesis) hypothesis)

Development of macroscopic manifestations reactions Development of macroscopic manifestations reactions (e.g. immunoprecipitation)(e.g. immunoprecipitation)

Ag – Ab reactions involving IgM are confined to the Ag – Ab reactions involving IgM are confined to the blood stream, while those of lower molecular weight blood stream, while those of lower molecular weight (IgG and IgE) can leave the vasculature and enter (IgG and IgE) can leave the vasculature and enter tissues tissues

Time required is hours to days for precipitin formation Time required is hours to days for precipitin formation leading to irreversible immunoprecipitatesleading to irreversible immunoprecipitates

LATTICE THEORYLATTICE THEORY

Lattice formation (visible Ag - Ab aggregates) Lattice formation (visible Ag - Ab aggregates) occurs when:occurs when:

– Ag is multivalent (contains more than 2 Ag is multivalent (contains more than 2 identical epitopes)identical epitopes)

– Cross-linking of Ags by specific Abs (2 or Cross-linking of Ags by specific Abs (2 or more antigen-binding sites)more antigen-binding sites)

– Molar ratios of epitopes and antigen-binding Molar ratios of epitopes and antigen-binding sites are optimal (zone of equivalence)sites are optimal (zone of equivalence)

Zone ofequivalence

LATTICE THEORYLATTICE THEORYZones of lattice formationZones of lattice formation– Far Ag excess (no ppt. formed; free Ag in Far Ag excess (no ppt. formed; free Ag in

supernatant) -- “postzone”supernatant) -- “postzone”

– Ag excess (sub-optimal ppt.; free Ag in spnt.)Ag excess (sub-optimal ppt.; free Ag in spnt.)

– Zone of equivalence (maximum ppt.; no Ag or Ab in Zone of equivalence (maximum ppt.; no Ag or Ab in spnt.)spnt.)

– Ab excess (sub-optimal ppt; Ab in spnt.)Ab excess (sub-optimal ppt; Ab in spnt.)

– Far Ab excess (no ppt; Ab in spnt.) -- “prozone”Far Ab excess (no ppt; Ab in spnt.) -- “prozone”

ZONES OF PRECIPITIN FORMATIONZONES OF PRECIPITIN FORMATION

Precipitin Curve

METHODS THAT DETECT Ag- Ab REACTIONSMETHODS THAT DETECT Ag- Ab REACTIONS

Primary Reactions:Primary Reactions:

- Immunofluorescence (IF)- Immunofluorescence (IF)

- Radioimmunoassay (RIA)- Radioimmunoassay (RIA)

- Enzyme immunoassay (EIA)- Enzyme immunoassay (EIA)

- Immunonephelometry (measures picogram to - Immunonephelometry (measures picogram to

nanogram quantities of analyte)nanogram quantities of analyte)

Secondary ReactionsSecondary Reactions

- Agglutination Techniques- Agglutination Techniques

- Precipitation Techniques ± Electrophoresis - Precipitation Techniques ± Electrophoresis

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

Precipitation in gelPrecipitation in gel

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

SINGLE VS. DOUBLE DIFFUSIONSINGLE VS. DOUBLE DIFFUSION

Single diffusionSingle diffusion– Supporting medium (gel) contains one Supporting medium (gel) contains one

reactant at a uniform concentrationreactant at a uniform concentration– Only the unknowns move through the Only the unknowns move through the

mediummedium

Double diffusionDouble diffusion– Gel is inert (contains no reactants)Gel is inert (contains no reactants)– Both Ag and Ab travel through the mediumBoth Ag and Ab travel through the medium

The region of equivalence

RADIAL IMMUNODIFFUSIONRADIAL IMMUNODIFFUSIONAb uniformly distributed in gel; Ag diffuses outward Ab uniformly distributed in gel; Ag diffuses outward from a well (single diffusion) from a well (single diffusion)

Ag- Ab complexes form as concentric rings around the Ag- Ab complexes form as concentric rings around the well at zone of equivalencewell at zone of equivalence

At a set time, ring diameters are measuredAt a set time, ring diameters are measured

[Ag] is directly proportional to the ring d[Ag] is directly proportional to the ring d22

Unknown value is determined by comparing to a 3-Unknown value is determined by comparing to a 3-standard curve standard curve

RADIAL IMMUNODIFFUSIONRADIAL IMMUNODIFFUSION

Standard Curve

Precipitin Rings A B C a b c

Standards Samples

RADIAL IMMUNODIFFUSIONRADIAL IMMUNODIFFUSIONFahey method (kinetic)Fahey method (kinetic)– Read at 18 hoursRead at 18 hours– Plot [std] vs. ring diameter on semi-log paperPlot [std] vs. ring diameter on semi-log paper

Mancini method (endpoint)Mancini method (endpoint)– Read at 48 or 72 hoursRead at 48 or 72 hours– Plot [std] vs. ring diameter squared on graph paperPlot [std] vs. ring diameter squared on graph paper

Results reliable only if the ring size is within the range Results reliable only if the ring size is within the range of the standards; if greater than highest std, dilute and of the standards; if greater than highest std, dilute and repeat testrepeat test

Used to measure IgM, IgG, C4,C3,transferrin, CRP, Used to measure IgM, IgG, C4,C3,transferrin, CRP, othersothers

OUCHTERLONY DOUBLE DIFFUSIONOUCHTERLONY DOUBLE DIFFUSION

Ag & Ab placed in wells cut into an agarose gel Ag & Ab placed in wells cut into an agarose gel (both reactants diffuse)(both reactants diffuse)

Precipitin line (or arc) indicates Ab has Precipitin line (or arc) indicates Ab has specificity for Agspecificity for Ag

Position of precipitin between wells depends on Position of precipitin between wells depends on MW and concentration of reactantsMW and concentration of reactants

3 possible patterns of reaction: identity, non-3 possible patterns of reaction: identity, non-identity, partial identityidentity, partial identity

OUCHTERLONY DOUBLE DIFFUSIONOUCHTERLONY DOUBLE DIFFUSION

Ouchterlony Plates Precipitin Patterns

ELECTROIMMUNOASSAY (ROCKET)ELECTROIMMUNOASSAY (ROCKET)

Electrophoresis hastens movement of Ag (placed in Electrophoresis hastens movement of Ag (placed in wells) through Ab -imbedded gel (single diffusion)wells) through Ab -imbedded gel (single diffusion)

Selected pH (8.6) keeps Abs at their isoelectric point; Selected pH (8.6) keeps Abs at their isoelectric point; they will not movethey will not move

Rocket-shaped precipitin bands will form at zone of Rocket-shaped precipitin bands will form at zone of equivalence (changes as reactants move)equivalence (changes as reactants move)

[Ag] proportional to length of rocket [Ag] proportional to length of rocket

Unknowns compared to standardsUnknowns compared to standards

ELECTROIMMUNOASSAY (ROCKET)ELECTROIMMUNOASSAY (ROCKET)

ELECTROIMMUNOASSAY (ROCKET)ELECTROIMMUNOASSAY (ROCKET)

May be used to quantitate plasma proteins May be used to quantitate plasma proteins such as coagulation factors, alpha-fetoprotein, such as coagulation factors, alpha-fetoprotein, C3, C4, CRP, haptoglobin C3, C4, CRP, haptoglobin

Compared with RID:Compared with RID:– fasterfaster– similar sensitivitysimilar sensitivity– requires electrophoretic equipment and more requires electrophoretic equipment and more

technological finessetechnological finesse

Largely replaced by immunonephelometryLargely replaced by immunonephelometry

IMMUNONEPHELOMETRYIMMUNONEPHELOMETRYAg + Ab Ag + Ab AgAb AgAb microscopic Ag - Ab complexes microscopic Ag - Ab complexes

Microcomplexes cause light moving through the Microcomplexes cause light moving through the suspending solution to scatter suspending solution to scatter

Nephelometer detects light scattered at a 90Nephelometer detects light scattered at a 90oo angle angle

Amount of light scattered at 90Amount of light scattered at 90oo is proportional to Ag - is proportional to Ag - Ab complexes formedAb complexes formed

Sensitive and quantitative technique used for Sensitive and quantitative technique used for measurement of many serum proteinsmeasurement of many serum proteins

IMMUNOELECTROPHORESIS (IEP)IMMUNOELECTROPHORESIS (IEP)

Electrophoresis and double diffusionElectrophoresis and double diffusion

2 stages2 stages– Proteins separated by electrophoresisProteins separated by electrophoresis– Antiserum placed in trough parallel to separated Antiserum placed in trough parallel to separated

proteins; all reactants diffuse in all directionsproteins; all reactants diffuse in all directions– Precipitin forms at zones of equivalencePrecipitin forms at zones of equivalence

Trough may be filled with simple or complex antisera Trough may be filled with simple or complex antisera yielding simple to complex patternsyielding simple to complex patterns

33

Immunoelectrophoresis of normal human serum.

IMMUNOELECTROPHORESIS (IEP)IMMUNOELECTROPHORESIS (IEP)Qualitative to semi-quantitativeQualitative to semi-quantitative

Serum, urine, or CSF may be analyzedSerum, urine, or CSF may be analyzed

Complex patterns may be difficult to interpretComplex patterns may be difficult to interpret

Useful to detect:Useful to detect:– missing proteinsmissing proteins– abnormal proteinsabnormal proteins– normal proteins in abnormal concentrationsnormal proteins in abnormal concentrations

Used to evaluate conditions such as multiple myelomaUsed to evaluate conditions such as multiple myeloma

Largely replaced by immunofixationLargely replaced by immunofixation

IMMUNOFIXATION IMMUNOFIXATION ELECTROPHORESIS (IFE)ELECTROPHORESIS (IFE)

Proteins that were separated by electrophoresis are Proteins that were separated by electrophoresis are exposed to Ab directly, instead of through diffusionexposed to Ab directly, instead of through diffusion

Steps:Steps:– Electrophoresis of protein mixture in gel (use Electrophoresis of protein mixture in gel (use

serum or urine samples)serum or urine samples)– Paper strips imbedded with specific Ab are “blotted” Paper strips imbedded with specific Ab are “blotted”

onto gel; Ags transfer to paper and bind to Absonto gel; Ags transfer to paper and bind to Abs– Strips washed (unbound material washes away)Strips washed (unbound material washes away)– Strips stained to reveal precipitin bandsStrips stained to reveal precipitin bands

IFEIFE

IMMUNOFIXATION IMMUNOFIXATION ELECTROPHORESIS (IFE)ELECTROPHORESIS (IFE)

Used to detect the presence of Igs in conditions Used to detect the presence of Igs in conditions like multiple myelomalike multiple myeloma

Fairly sensitive - Ab is highly specific, Fairly sensitive - Ab is highly specific, electrophoresis leaves Ag isolated and electrophoresis leaves Ag isolated and accessibleaccessible

Faster and easier to interpret than IEPFaster and easier to interpret than IEP

Only 1 Ab may be used per stripOnly 1 Ab may be used per strip

WESTERN BLOTTINGWESTERN BLOTTINGSimilar to IFE but the unknown is Ab rather than AgSimilar to IFE but the unknown is Ab rather than Ag

Steps:Steps:– Separation of complex antigenic material (eg., viral Separation of complex antigenic material (eg., viral

proteins) by electrophoresisproteins) by electrophoresis– Separated components transferred from gel to Separated components transferred from gel to

nitrocellulose paper by “blotting”nitrocellulose paper by “blotting”– Unknown (or control) sera (which may have Abs) Unknown (or control) sera (which may have Abs)

incubated with paper strips; Ag - Ab complexes ppt. incubated with paper strips; Ag - Ab complexes ppt. at site of transferat site of transfer

– Strips washed; staining reveals complexesStrips washed; staining reveals complexes

WESTERN BLOTTINGWESTERN BLOTTING

42The Western blot procedure.

FLOCCULATIONFLOCCULATIONImmunoprecipitation (or agglutination) of Immunoprecipitation (or agglutination) of insolubleinsoluble particles particles

Characterized by very sharp pro- and Characterized by very sharp pro- and postzonespostzones

No precipitin formed in zones of Ab or Ag No precipitin formed in zones of Ab or Ag excess, only in zone of equivalenceexcess, only in zone of equivalence

Clinically important examples, VDRL and RPR Clinically important examples, VDRL and RPR tests (screening tests for syphilis)tests (screening tests for syphilis)

FLOCCULATION VS. IMMUNOPRECIPITATIONFLOCCULATION VS. IMMUNOPRECIPITATION

Flocculation Tests

- VDRL (Venereal Disease Research Lab.) testVDRL (Venereal Disease Research Lab.) test- RPR (Rapid Plasma Reagin) testRPR (Rapid Plasma Reagin) test

AgglutinationAgglutinationTiterTiter

Zeta potentialZeta potential

Types of AgglutinationTypes of Agglutination - Direct agglutination or hemagglutination- Direct agglutination or hemagglutination - Indirect (passive) agglutination or hemagglutination- Indirect (passive) agglutination or hemagglutination - Agglutination or hemagglutination inhibition- Agglutination or hemagglutination inhibition

The Coombs testThe Coombs test - Direct- Direct - Indirect- Indirect

Agglutination ReactionsAgglutination 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 thehighest dilution of serum showing agglutination.

1:100 1:200 1:400

Titer

Agglutination inhibition

Hemagglutination Inhibition TestHemagglutination Inhibition TestTo Detect AntibodiesTo Detect Antibodies (Rubella) (Rubella)

- Serum (Ab)+ HA +RBCs=- Serum (Ab)+ HA +RBCs= No Hemagglutination No Hemagglutination

= = Positive TestPositive Test

- Serum (No Ab)+ HA + RBCs - Serum (No Ab)+ HA + RBCs =Hemagglutination =Hemagglutination ==Negative TestNegative Test

To Detect AntigenTo Detect Antigen (HBsAg) (HBsAg)

- Serum (HBsAg) +Anti HBsAG + HBsAg coated RBCs- Serum (HBsAg) +Anti HBsAG + HBsAg coated RBCs = = No Hemagglutination No Hemagglutination = = Positive TestPositive Test

- Serum (No HBsAg)+ Anti HBsAG + HBsAg coated - Serum (No HBsAg)+ Anti HBsAG + HBsAg coated RBCsRBCs == Hemagglutination Hemagglutination ==Negative TestNegative Test

Use of Labels in Ag – Ab ReactionsUse of Labels in Ag – Ab ReactionsImmunoassaysImmunoassays

- Radioimmunoassay (RIA)- Radioimmunoassay (RIA)

- Enzyme Immunoassys (EIA)- Enzyme Immunoassys (EIA)

ImmunofluorescenceImmunofluorescence (IF) (IF)

-- Direct IFDirect IF

- Indirect IF- Indirect IF

Flow cytometry and Cell Sorting (FACS)Flow cytometry and Cell Sorting (FACS)

Immunologic Tests4) 4) RadioimmunoassayRadioimmunoassay (RIA)– a very sensitive test; (RIA)– a very sensitive test;

used for measuring hormones, serum proteins, drugs, used for measuring hormones, serum proteins, drugs, etc. at low concentrations (etc. at low concentrations (≤ 0.001ug/ml)≤ 0.001ug/ml)

measures “measures “competitive binding”competitive binding” of radiolabelled Ag of radiolabelled Ag + unlabelled (test) Ag to high affinity Ab+ unlabelled (test) Ag to high affinity Ab

ELISA

ELISA testsELISA 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 Direct and indirect ImmunofluorescenceImmunofluorescence

ImmunoprecipitationImmunoprecipitationProvides a quick and Provides a quick and sensitive test for finding sensitive test for finding proteins/Ag’s especially proteins/Ag’s especially in low concentrationsin low concentrations

Binds Ab to synthetic Binds Ab to synthetic bead support bead support centrifugedcentrifuged

Or 2Or 2°° Ab with bead or Ab with bead or magnetic bead and magnetic bead and collect by magnetismcollect by magnetism

Distribution of selected markers on some leukemia cell types → Immunophenotyping using

“flow cytometry & mAb”

Sensitivity of various immunoassays