measure the concentration of an analyte usually
antigens of infectious agent or antibodies against
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them in bodily fluids.In this technique, the antigen or antibody from the
sample tested is immobilised from the liquid phase
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on to solid phase, typically wells of microtitre plate.
Principle of ELISA
Based on basic immunological response i.e.
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formation of antibody-antigen complex and
its detection by the reaction with another
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antibody, which is labelled with a reporterenzyme producing a signal in the form of
change in color when specific substrate is
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added.
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Reagent
composition
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Coating Buffer0.01 M Phosphate Buffer +0.1M NaCL
Diluting /Washing Buffer
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Phosphate Buffer + NaCL +0.1% Tween 20
Blocking Buffer
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BSAEnzyme
HRP,ALP
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Chromogenic Substrate
TMB, PNPP
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Stop Solution0.5 M Sulfuric Acid
Double Antibody Sandwich ELISA
DAS ELISA also called direct ELISA is probably the most
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widely used immunochemical technique in diagnostics.
The principle is that antigen is immobilised on a solid
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phase by a primary coating antibody and detected with asecond antibody that has been labelled with a marker
enzyme
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The antigen creates a bridge between the two antibodies
and the presence of the enzyme causes a color change in
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the chromogenic substrateTriple Antibody Sandwich ELISA
Also known as indirect ELISA, is a method often used to
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identify antibodies against pathogens in patient blood todiagnose infection.
As an example of a diagnostic test, HBV capture antibody
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is bound to the well of microtitre plate and coat protein
from the virus (the antigen) is added.
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After incubation and washing, patient serum is added,which ,if it contains antibodies, reacts with antigen.
Anti-human antibody conjugate to an enzyme marker is
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added to identify the samples which are positive for HBV
antigen
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The test works well for the diagnosis of HBV infection andis also used to ensure that blood donations given for
transfusion are free from this virus.
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Competitive ELISA
The principle is based on the competition between the natural unlabelled
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antigen to be tested AND a labelled (enzyme-conjugate) form of the antigen .The test sample and a defined amount of enzyme-conjugated antigen are
mixed together and placed in to antibody-coated wells of a microtitre plate
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The antigen and the conjugated derivative compete for available spaces on the
coated antibody layer
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The more natural unlabelled antigen present, the more it will displace thelabelled form, leading to a reduction in enzyme bound to plate
Increased serum antigen results in reduced binding of the antigen-enzyme
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conjugate with the capture antibody producing less enzyme activity and
reduced color formation.
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This form of ELISA is routinely use for testing blood
samples for thyroxine
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Competitive ELISA provide an accurate measure ofthe circulating level of the hormone compared to
standard curve of known dilutions
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APPLICATIONSScreening donated blood for evidence of virus
contamination by
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HIV-1 and HIV-2(presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies)
Hepatitis B (presence of both antigen and antibodies)
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Measuring hormone levels of HCG,LH ,TSH,T3 and T4
Detecting infections like HIV, syphilis and chlamydia
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or Hepatitis B and C.