Download MBBS ELISA Lecture PPT

ELISA

Enzyme- linked immunosorbent assay (ELISA) is a

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common laboratory technique which is used to

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 reporter

enzyme 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 Buffer

0.01 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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BSA

Enzyme

HRP,ALP

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Chromogenic Substrate

TMB, PNPP

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Stop Solution

0.5 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 a

second 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 substrate

Triple 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 to

diagnose 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 and

is 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 the

labelled 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 of

the circulating level of the hormone compared to

standard curve of known dilutions

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APPLICATIONS

Screening 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.