Immunoelectrophoresis

Immunoelectrophoresis

1. Dr. J. P. Saranraj
IMMUNOELECTROPHORESIS

2. IMMUNOELECTROPHORESIS
ImmunoelectrophoresiswasfirstcoinedbyGrabarand
Williamsin1953.
Immunoelectrophoresisisageneralnameforanumberof
biochemicalmethodsforseparationand
characterizationof proteins based
onelectrophoresisandreactionwithantibodies.
Immunoelectrophoresisisthecombinationof
Immunodiffusion (Mancini’sSingleRadial
ImmunodiffusionandOuchterlonyDoubleDiffusion)and
Electrophoresis.

3. Single Radial Immunodiffusion

4. Double Immunodiffusion

5. Electrophoresis

6. IMMUNOELECTROPHORESIS
InImmunoelectrophoresis,theantigenmixtureisfirst
electrophoresedtoseparateitscomponentsbycharge.
Troughsarethencutintotheagargelparalleltothe
directionoftheelectricfield,andantiserumisaddedtothe
troughs.
Antibodyandantigenthendiffusetowardeachotherand
producelinesofprecipitationwheretheymeetin
appropriateproportions.
ImmunoelectrophoresisisastrictlyQualitativetechnique
thatonlydetectsrelativelyhighantibodyconcentrations
(greaterthan100g/ml),itutilityislimitedtothedetection
ofquantitativeabnormalitiesonlywhenthedeparturefrom
normalisstriking,asinimmunodeficiencystates.

7. IMMUNOELECTROPHORESIS

8. Applications of Immunoelectrophoresis
Immunoelectrophoresisisusedinclinicallaboratoriesto
detectthepresenceorabsenceofproteinsintheserum.
DetectionofImmunodeficiency(InImmunodeficiency
sample,noprecipitinbandisformedwithparticular
antigen).
DetectionofOverproductionofSerumproteins(Albumin,
ImmunoglobulinandTransferrin).
DetectionofdeficiencyinComplement.
UsedtoidentifynormalandabnormalproteinsinUrineor
Serum.
TestingthepurityofAntigen.

9. ROCKET ELECTROPHORESIS
ArelatedQuantitativetechnique,Rocket
electrophoresis,doespermitmeasurementofantigen
levels.
AlsocalledasLaurellTechniqueorOnedimension
electroimmunodiffusion.
ItiscalledasanadaptionofSRIDandmorerapidthan
SRID.
InRocketelectrophoresis,anegativelychargedantigen
iselectrophoresedinagelcontainingantibody.
Theprecipitateformedbetweenantigenandantibodyhas
theshapeofaRocket(measured),theheightofwhich
isproportionaltotheconcentrationofantigeninthewell.

10. Onelimitationofrocketelectrophoresisistheneedforthe
antigentobenegativelychargedforelectrophoretic
movementwithintheagarmatrix.Someproteins,
immunoglobulinsforexample,arenotsufficientlycharged
tobequantitativelyanalyzedbyRocketelectrophoresis.