ELISA
ELISA
Principle : Ag-Ab binding is detected using enzyme
which converts a colorless substrate (chromogen) to
a colored product
Evolved from radioimmunoassay (RIA), a technique using
radioactively-labeled antigens or antibodies
Used to quantify any antigen/antibody
Hormone
Peptide
Protein
Antibody
Procedure
Generally performed in a 96 well plate (ELISA
plate). The well is coated with the reagent
Antibody (or Antigen)
Primary Incubation: Sample (serum, urine
etc) is added to the well. Antigen & Antibody are
allowed to interact
Washing: Removes non specifically bound
Antibody or antigen
Secondary incubation: A second antibody
directed against the Fc portion of the primary Ab is
added. It is labelled with an indicator enzyme
(e.g. Alkaline phosphatase, Horse radish peroxidase)
Washing: Removes non specifically bound
Secondary antibody
Detection: Suitable substrate (chromogen) is
added according to the enzyme used for
development of color (ALP substrate – PNPP, p-
nitrophenyl phosphate; HRP substrate – TMB,
3,3',5,5'-tetramethylbenzidine)
Washing Secondary antibody
substrate Enzyme
Washing
Colored
product
Proportio
nal to
Primary antibody
Antigen of interest
Different antigen in
Types of ELISA
Non- Indire Direc Sand
competitiv ct t wich
e ELISA ELISA ELISA ELISA
Competiti
ve ELISA
Direct ELISA
ELISA in which only a labeled
primary antibody is used
The well is directly coated with the
sample
Enzyme linked primary antibody is
applied to the plate
Washing : After this wash, only the
antibody-antigen complexes remain
attached
Apply a substrate which is
converted by the enzyme to elicit a
chromogenic signal
Indirect ELISA
ELISA in which the antigen is bound by the primary antibody
which then is detected by a labeled secondary
antibody (Two step binding)
Antigen is added to plate.
Suitable primary antibody is
added.
Secondary antibody – HRPO is
then added which recognizes and
binds to primary antibody.
TMB substrate is added, is
converted to detectable form.
Sandwich ELISA
Uses two antibodies specific to the antigen to capture
or "sandwich” antigens in the well for detection
1. Capture antibody coated on the plate to bind
the antigen of interest
2. Detection antibody binds a different epitope
of the same antigen
3.
The detection antibody can either be bound by a
secondary antibody-enzyme conjugate, or the detection
antibody itself is enzyme-conjugated
Plate is coated with suitable capture antibody.
Sample is added to plate so antigen is bound by capture
antibody.
A suitable biotin labeled detection antibody is added
to plate.
Enzyme HRPO is added and binds the biotin labeled detection
antibody.
TMB substrate is added and converted by HRPO to colored
product.
Procedure
Competitive ELISA
Antigen in the sample competes for limited antibody
binding sites with antigen conjugated to a reporter
enzyme
Unlabelled Ag – Patient sample
Labeled Ag – Reagent
This produces an inverse relationship between
antigen concentration and colored
product (more the bound fraction of Antigen is
labelled, higher the color)
Competitive ELISA
Solid phase coated with
antibody
Add unknown amount of
unlabeled antigen and known
amount of labeled antigen
Free and labeled antigen are
captured
Color formation by oxidation of
substrate
into a colored compound
The enzyme activity measured is proportional to the proportion of labeled antigen in the
mixture of labeled and unlabeled antigen.
Comparison between various types
of ELISA
Direct Indirect Sandwich Competitive
Quantitative ELISA
Known concentrations of antigen are used to produce a
standard curve
Concentration of unknown samples is measured by
comparing with the linear portion of the standard curve.
Applications of ELISA
Screening donated
blood for evidence of
viral contamination by HIV-1 and HIV-2 (anti-p24 antibodies)
Hepatitis C (antibodies)
Hepatitis B (antibodies, viral antigen - HBsAg)
Measuring hormone
levels
HCG (as a test for pregnancy)
LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)
Detecting infections Sexually-transmitted infections (STI) – HIV,
syphilis, Chlamydia
Hepatitis B and C
Toxoplasma, Rubella, CMV, Herpes (TORCH) -
pregnancy
Detecting illicit drugs
THC, Cocaine, Methamphetamine
Detecting allergens in
food and house dust
Pollen, Insect
