Ag-Ab Reactions

Antigen - Antibody reactions

1. Dr. Pulipati Sowjanya
Professor & Head
Dept. of Pharm. Biotechnology
Vignan Pharmacy College
Vadlamudi, Guntur (Dt)

2. INTRODUCTION
DEFINITION:Antigen-antibodyreaction,isaspecificchemical
interactionbetweenantibodiesproducedbyBcellsoftheWBCand
antigensduringimmunereaction.
Thisreactionoccursinthreestages.
1.Theprimarystage-rapid,reversible.
2.Thesecondstage-precipitation,agglutination,lysisofcells.
3.Thetertiarystage–destructionofantigens.

3. GENERAL FEATURES
✓Thereactionisspecific.
✓Thereisnodenaturationoftheantigen–antibody
duringreaction.
✓Thecombinationisfirmbutreversible.
✓Bothantigen&antibodiesaremultivalent.
✓Bothantigen&antibodiesparticipateintheformation
ofagglutinatesorprecipitates.

4. MEASUREMENT
➢Antigen-antibodyparticipatinginthereactionmaybemeasured
intermsofmassorunitsortiter.
➢Theantibodytiteristhehighestdilutionofserumthatgivesan
observablereactionwiththeantigenintheparticulartest.It
influencedbythenatureandquantityoftheantigen.
➢Antigensmayalsobetitratedagainstsera.

5. Thetwoimportantparametersofserologicaltestare:
SENSITIVITY:Itistheabilitytodetectevenveryminute
quantitiesofantigenorantibodies.Whenatestishighlysensitive,
falsenegativeresultswillbeabsent.
SPECIFICITY:Itistheabilitytodetectreactionbetween
homologousantigen&antibodiesonly.Inahighlyspecifictest,false
positiveresultswillbeabsent.

6. TYPES OF ANTIGEN-ANTIBODY REACTIONS
PRECIPITATIONREACTIONS:
DEFINITION:-Whenasolubleantigencombineswithits
antibodyinthepresenceofanelectrolyteatasuitable
temperature(37
o
C)andP
H
(7.4)itformsanantigen–antibody
complexintheformofaninsolubleprecipitate.
▪Theantigen-“precipitinogen”andantibody-“precipitin”.
▪Iftheprecipitateremainssuspendedasfloccules,thereactionis
knownas“flocculation”.

7. MECHANISM
❖Marrack(1934)proposedthelatticehypothesistoexplain
themechanismofantigen–antibodyreaction.
❖Accordingtothishypothesismultivalentantigencombine
withbivalentantibodiesinvaryingproportions,dependon
theantigen-antibodyratioinreactionmixture.

9. APPLICATIONS
a.Thesearehighlysensitiveforthedetectionofantigen.
b.Diagnosisofinfectiousdisease.
c.Forensicmedicineintheidentificationofblood&
semenstainsonweapons&clothingorinothercriminal
investigations.
d.Detectionoffoodadulterants.
e.Detectionofunknownantibody.

10. TYPES OF PRECIPITATION REACTIONS
Single/ double diffusion in
one dimension.
PRECIPITATION
TESTS
IN LIQUIDS IN GELS
Single / double diffusion in
two dimensions.
Counter-current & rocket
electrophoresis.
Ring test
Slide test
Tube test

11. PRECIPITATION IN LIQUIDS
RING TEST:
▪Inapositivecasearingofwhite
precipitateisformedatthejunction
oftwoliquids.
Ex:Ascolisthermoprecipitintest

12. SLIDE TEST:-
Adropeachofantigenandantibodyareplacedon
aslideandmixedbyrotation,flocculesappear.
Ex:testforsyphilis
TUBETEST-:
Theantigenandantibodyaretakeninatesttube
mixed,incubatedandthenobservedforprecipitation
Ex:Kahntestisatubeflocculationtestusedforthe
diagnosisofsyphilis.

13. PRECIPITATIONIN GELS
Gelprecipitationtestsaremoreadvantageousthanthe
liquidprecipitationtest.
Thebandsarestable,canbestained&preserved.
Thegelshasgotminuteporesthroughwhichtheantigens
andantibodiesmigrate&formbandswheretheymeetin
optimumproportion.
Sinceeachantigen–antibodyreactiongivesrisetobandof
precipitation,thenumberofantigensinthereactionmixture
canbedetected.

14. TYPES
1.SINGLEDIFFUSIONINONEDIMENSION:
ItisalsocalledasOudinmethod.
Theantibodyisincorporatedinagargelin
atesttubeandtheantigensolutionislayered
overit.
USES:
Thenumberofbandsindicatesthenumberof
differentantigenspresent.

15. DOUBLE DIFFUSION IN ONE DIMENSION:
ItisalsocalledasOakly–Fulthorpe
procedure.
Thismethodseparatesantigenandantibody
solutionwithalayerofclearagar.
USES:
Thistestisusedtodeterminethenumberof
antigensinamixture.

16. 3.SINGLEDIFFUSIONINTWODIMENSIONS:
Itisalsocalledasradialimmunodiffusion.
Antiserumisincorporatedinagargelat56
0
Candpouredonaglass
slideorintopetridishes.
Thegelisallowedtoset.WellsarecutandAntigenisadded.
Diameteroftheringgivesanestimateoftheconcentrationof
theantigen.

17. 4.Doublediffusionintwodimensions:
•ThismethodisalsoknownasOuchterlonymethod.
•Antiserumisplacedinthecentralwallandantigeninthe
surroundingwells.
USES:
•Used to detect & compare difference between antigen & antibody.
•In the diagnosis of bacterial, viral, fungal and parasitic infection.

19. ELECTRO IMMUNODIFFUSION
•It was discovered by Graber and Williams, 1953.
1.Counter –current immunoelectrophoresis: This involves
simultaneous electrophoresis of Ag & Ab in gel in opposite direction &
form a precipitation line within few minutes is called “cross-over”.
Antibodiestendtomigratetowardscathodewhilemostantigenic
protiensmovetowardsanode.
USES:
❖Detection of antigen in various body fluids.
❖Diagnosis of bacterial, viral, fungal infections
Ag Ab
- +

20. 2. Rocket electrophoresis
•It is a simple technique described by Laurell
in 1965.
•Antibody is mixed with agarose gel and
poured on a slide.
•Increasing concentration of antigen is added
to wells.
•Antigen moves toward the anode.
•Based on the height of the rocket ,
concentration of antigen is estimated.
USES:
•Estimation of proteins and other antigens in
various body fluids.
Direction
Of current
Increasing concentrations of
antigen in wells
cathode
anode

22. Immunoelectrophoresis
•Method
–Agsare separated by electrophoresis
•Interpretation
–Precipitin arc represent individual antigens
Ag
-+
Ag
Ab
Ag
Ab
–Ab is placed in trough cut in the agar

23. AGGLUTINATION REACTION
•Whenaparticulateantigenismixedwithitsantibodyinthe
presenceofanelectrolyteatasuitabletemperatureandP
H
.
•Antigen-agglutinogenandantibody-agglutinin.
TYPES:-1.SlideTest:
A drop of antiserum is added to the suspension & mixed.
The clumping together of the particles & clearing of the drop
within few seconds indicate positive results.
USES:
Blood grouping and cross matching can be detected.

24. 2.TUBE TEST
Serum is diluted in saline serially by double dilution in a
series of test tubes.
An equal volume of standard antigen suspension is added
to all the tubes.
Highest dilution of serum at which agglutination occurs is
recorded as antibody titre.
USES:
Widal test for diagnosis of enteric fever.
Tube agglutination for brucellosis.
Weil-Felix test for Typhus fever.

27. MicrotitrationAgglutination Tests
Thesetestsaredoneinmicrotitreplates
1.Indirectpassivehaemagglutinationtest:
Carrierparticle:Agiscoatedonredcells.Turkeyredcellsare
oftenselectedasredcarrierredcells.Theformalinfixedred
cellsaretreatedwithtannicacidtomaketheantigenadhere.
Thesearecalledsensitizedcells.
Procedure:Seriallydilutedpatientserumismixedwithsensitized
cells.
Result:Positiveresultshowsagglutinationofredcells

28. MicrotitrationAgglutination Tests
2.Haemagglutinationinhibitionantibody(HAI)test:
detectsantibodiesagainstviruses(arbo,influenza,measles,rubella,
orthomyxo&paramyxo)
Procedure:
❑Patientserumistreatedwithkaolinorheparin-magnesiumchloride(to
removenonspecificinhibitors)
❑Theserumisallowedtoreactwithasuspensionofknownviral
antigens
Result:Ifantibodiestovirusarepresent,itwillcoatthehaemagglutinins
ontheviralparticlesandwillformcomplexeswhichwillblockthe
bindingsitesontheviralsurfacepreventinghaemagglutination.

30. MicrotitrationAgglutination Tests
3.Reversepassivehaemagglutination(RPHA)test:
❑Thistechniqueutilizesstabilizedredbloodcellscoatedwith
specificantibody.
❑Ifthecorrespondingantigenispresent,theredcellswill
agglutinate.
❑Itisusefulfornon-agglutinatingviruses.

31. LATEX AGGLUTINATION TEST:
➢ Latexisaninertparticleofmineral
originwithauniformdiameterof0.08–1
micron.
➢Antigenmoleculesareadsorbedonto
thesurfaceofparticleslikebentoniteclay
orlatex.
USES:
•C-Reactive Protein test.
•Antistreptolysin O test.

32. Complement fixation test
•It tests for the presence of either specific antibodyor specific
antigen in a patient's serum.
•It uses sheep red bloodcells (sRBC), anti-sRBCantibody and
complement, plus specific antigen or specific antibody.
•If either the antibody or antigen is present in the patient's serum,
then the complement is completely utilized, so the sRBCsare not
lysed. But if the antibody (or antigen) is not present, then the
complement is not used up, so it binds anti-sRBCantibody, and
the sRBCsare lysed.
•The is one form of complement fixation test

34. Theneutralizationreactionarethereactionsoftheantigen–
antibodythatinvolvestheeliminationofharmfuleffectsofbacterial
exotoxinsoravirusbyspecificantibodies.Theneutralizingsubstance
i.e.,antibodiesareknownasantitoxins.
USES:
1.Diagnosisofviralinfection:
2.Shicktest
NEUTRALISATION TEST

35. NEUTRALISATION TEST

36. IMMUNOFLUORESENCE
Fluorescenceisapropertyexhibitedbycertainsubstanceswhich
absorblightraysofoneparticularwavelengthandthenreleasethe
absorbedenergybyemittingraysofdifferentwavelength.
Theseareof2types:
1.DIRECT:AbtotissueAgislabeledwithfluorochrome
USES:
Toidentifymicroorganismspresent
inclinicalspecimens
Ag
Fluorochrome
Labeled Ab
Tissue Section

37. 2.INDIRECT:
ToKnowntissueAgattachedonslideunknownserumisadded
Fluorochrome-labeledanti-Igisusedtodetectbindingofthefirst
Ab.
USES:
DiagnosisofSyphilis.
Ag
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled
Ab

38. .
.
.
DIRECT TEST IMMUNOFLUORESENCE
antigen
fluorescent conjugated -
antibody
ANTIGEN –ANTIBODY COMPLEX
INDIRECT TEST -
IMMUNOFLUORESCENCE

39. RADIOIMMUNO ASSAY:
Themostsensitivetechniquefordetectingantigenorantibody.
Firstdevelopedbytwoendocrinologist,S.ABersonandRosalyn
Yalowinin1960todeterminelevelsofinsulin–antiinsulincomplexes
indiabetics.
1.LiquidPhaseRIA:
❑Increaseamountofantigen(unlabeled)ofunknownconcentrationis
added.
❑Theamountoflabeledantigeninsolutionismeasured&the
constructionofunlabeledantigencanbedetermined.
2.SolidPhaseRIA:
ItissimpleeasyinhandlingascomparedtoliquidPhaseRIA.

41. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
•Uses an enzyme system to show the specific combination of
antigen antibody
•An enzyme labeled or linked to a specific antigen
•A substrate
•A color reader
•Double antibody technique to detect and assay antigen
•Indirect technique to Assay and antibody
TYPES
a.Indirect ELISA
b.Sandwich ELISA
c.Competitive ELISA

42. •Done on Polyvinyl microtitreplate
Enzyme substrate color
•Horse radisho-phenyl-diamine red/
peroxidase dihydrochlorideorange
•Alkaline p-nitro phenyl yellow
phosphatasephosphatase
USES:
a.Detection of viral antigens like Rota virus, Hepatitis.
b.Detection of hormones & toxins.
c.Pregnancy test.
Detection of antibodies in various bacterial & viral infection.
Bacterial –Salmonella, Vibriocholera, Haemophilus.
Viral -Rubella , HIV, Herpes simplex, Hepatitis B.
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)