Download MBBS ELISA Lecture PPT

Download MBBS (Bachelor of Medicine and Bachelor of Surgery) Latest ELISA Lecture PPT


ELISA

Enzyme- linked immunosorbent assay (ELISA) is a

common laboratory technique which is used to

measure the concentration of an analyte usually
antigens of infectious agent or antibodies against

them in bodily fluids.

In this technique, the antigen or antibody from the

sample tested is immobilised from the liquid phase

on to solid phase, typically wells of microtitre plate.
Principle of ELISA

Based on basic immunological response i.e.

formation of antibody-antigen complex and

its detection by the reaction with another

antibody, which is labelled with a reporter

enzyme producing a signal in the form of

change in color when specific substrate is

added.




Reagent

composition

Coating Buffer

0.01 M Phosphate Buffer +0.1M NaCL

Diluting /Washing Buffer

Phosphate Buffer + NaCL +0.1% Tween 20

Blocking Buffer

BSA

Enzyme

HRP,ALP

Chromogenic Substrate

TMB, PNPP

Stop Solution

0.5 M Sulfuric Acid
Double Antibody Sandwich ELISA
DAS ELISA also called direct ELISA is probably the most

widely used immunochemical technique in diagnostics.

The principle is that antigen is immobilised on a solid

phase by a primary coating antibody and detected with a

second antibody that has been labelled with a marker

enzyme

The antigen creates a bridge between the two antibodies

and the presence of the enzyme causes a color change in

the chromogenic substrate

Triple Antibody Sandwich ELISA
Also known as indirect ELISA, is a method often used to

identify antibodies against pathogens in patient blood to

diagnose infection.

As an example of a diagnostic test, HBV capture antibody

is bound to the well of microtitre plate and coat protein

from the virus (the antigen) is added.

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

added to identify the samples which are positive for HBV

antigen

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.

Competitive ELISA

The principle is based on the competition between the natural unlabelled

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

The antigen and the conjugated derivative compete for available spaces on the

coated antibody layer

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

conjugate with the capture antibody producing less enzyme activity and

reduced color formation.


This form of ELISA is routinely use for testing blood

samples for thyroxine

Competitive ELISA provide an accurate measure of

the circulating level of the hormone compared to

standard curve of known dilutions
APPLICATIONS

Screening donated blood for evidence of virus

contamination by

HIV-1 and HIV-2(presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies)

Hepatitis B (presence of both antigen and antibodies)

Measuring hormone levels of HCG,LH ,TSH,T3 and T4

Detecting infections like HIV, syphilis and chlamydia

or Hepatitis B and C.

This post was last modified on 30 November 2021