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