Precipitation Reaction

Precipitation Reaction

1. Dr. P. Saranraj
Head
Department of Microbiology
Sacred Heart College (Autonomous)
Tirupattur –635 601
Tamil Nadu, India
Mobile: [phone]; E.mail:
[email protected]
PRECIPITATION REACTION

2. PRECIPITATION REACTION
Precipitationreactionsarebasedonthe
interactionofantibodiesandantigens.Inthis
reaction,twosolublereactantsthatcome
togethertomakeoneinsolubleproduct,the
precipitate.
Anyantibodywhichreactswithanantigento
formaprecipitateiscalledPrecipitin.
Precipitationassaysareperformedinsemi-solid
mediasuchasAgarorAgarosewhereantibodies
andantigenscandiffusetowardoneanotherand
formavisiblelineofprecipitation.

3. Precipitationreactionsareserologicalassaysfor
thedetectionofimmunoglobulinlevelsfrom
theserumofapatientwithinfection.
Precipitationreactionsarewidelyusedin
MedicineforanalysisofHormones,Enzymes,
ToxinsandImmunesystemproducts.
SomeoftheexamplesofPrecipitationreactions
are1)CapillaryTubePrecipitation(RingTest),2)
Ouchterlony Double Immunodiffusion
(ImmunodiffusionorAgargelImmunodiffusionor
PassiveDoubleImmunodiffusion),3)Single
RadialImmunodiffusion(SRID)(Mancini
Method),4)Immunoelectrophoresis(IEP),5)
Rocket electrophoresis and 6)
Counterimmunoelectrophoresis(CIEP).

4. Ouchterlony Double Immunodiffusion
OuchterlonyDoubleImmunodiffusion(ODD)is
alsocalledasImmunodiffusionorAgargel
Immunodiffusion orPassive Double
Immunodiffusion.
ODDmethodwasusedinthedetection,
identificationandquantificationofantibodies
and antigens, such
asImmunoglobulinsandextractablenuclear
antigens.
Thetechniqueisnamed afterOrjan
Ouchterlony,theSwedishphysicianwho
inventedthetestin1948.
InODDmethod,thepatternoflinesthatform
canbeinterpretedtodeterminetherelationship

5. Ouchterlony Double Diffusion

6. Single Radial Immunodiffusion (SRID)
RadialImmunodiffusionorMancinimethod,
ManciniImmunodiffusionorSingleRadial
Immunodiffusion(SRID).
SRIDisanImmunodiffusiontechniqueusedto
determinethequantityorconcentrationof
anantigeninasample.
InSRIDmethodtheAntibodyisincorporatedinto
theAgarosegelwhereastheAntigendiffuses
intoitinaradialpattern.
SRIDisanImmunodiffusiontechniqueusedto
detecttheconcentrationofantigenby
measuringthediameteroftheprecipitinring
formedbytheinteractionoftheantigenandthe
antibodyatoptimalconcentration.

7. Single Radial Immunodiffusion

8. Electrophoresis

9. IMMUNOELECTROPHORESIS
Immunoelectrophoresiswasfirstcoinedby
GrabarandWilliamsin1953.
Immunoelectrophoresisisageneralnamefora
numberofbiochemicalmethodsforseparation
andcharacterizationofproteinsbased
on electrophoresis and reaction
withantibodies.
Immunoelectrophoresisisthecombinationof
Immunodiffusion(Mancini’sSingleRadial
ImmunodiffusionandOuchterlonyDouble
Diffusion)andElectrophoresis.

10. IMMUNOELECTROPHORESIS
InImmunoelectrophoresis,theantigenmixtureis
firstelectrophoresedtoseparateitscomponents
bycharge.
Troughsarethencutintotheagargelparallelto
thedirectionoftheelectricfield,andantiserumis
addedtothetroughs.
Antibodyandantigenthendiffusetowardeach
otherandproducelinesofprecipitationwhere
theymeetinappropriateproportions.
ImmunoelectrophoresisisastrictlyQualitative
techniquethatonlydetectsrelativelyhigh
antibodyconcentrations(greaterthan100g/ml),
itutilityislimitedtothedetectionofquantitative
abnormalitiesonlywhenthedeparturefrom
normalisstriking,asinimmunodeficiencystates.

11. IMMUNOELECTROPHORESIS

12. Applications of Immunoelectrophoresis
Immunoelectrophoresisisusedinclinical
laboratoriestodetectthepresenceorabsenceof
proteinsintheserum.
Detection of Immunodeficiency (In
Immunodeficiencysample,noprecipitinbandis
formedwithparticularantigen).
DetectionofOverproductionofSerumproteins
(Albumin,ImmunoglobulinandTransferrin).
DetectionofdeficiencyinComplement.
Usedtoidentifynormalandabnormalproteinsin
UrineorSerum.
TestingthepurityofAntigen.

13. ROCKET ELECTROPHORESIS
ArelatedQuantitativetechnique,Rocket
electrophoresis,doespermitmeasurementof
antigenlevels.
AlsocalledasLaurellTechniqueorOne
dimensionelectroimmunodiffusion.
ItiscalledasanadaptionofSRIDandmore
rapidthanSRID.
InRocketelectrophoresis,anegativelycharged
antigeniselectrophoresedinagelcontaining
antibody.
Theprecipitateformedbetweenantigenand
antibodyhastheshapeofaRocket
(measured),theheightofwhichisproportional
totheconcentrationofantigeninthewell.

14. Onelimitationofrocketelectrophoresisisthe
needfortheantigentobenegativelychargedfor
electrophoreticmovementwithintheagarmatrix.
Someproteins,immunoglobulinsforexample,
arenotsufficientlychargedtobequantitatively
analyzedbyRocketelectrophoresis.