ELISA

Enzyme-linked immunosorbent assay

DEFINITION
Rapid test used for detecting antibody (Ab) against
viruses, bacteria and other materials or antigen (Ag)
Antibodies are made in response to infection or harmful
substances called antigen
DIAGOSTIC FUNCTION
HIV, which causes AIDS
Lyme disease
Pernicious anemia
Rocky Mountain spotted fever
Rotavirus
Squamous cell carcinoma
Syphilis
Toxoplasmosis
Varicella-zoster virus, which causes chickenpox and shingles
Zika virus
MATERIAL
1. ELISA plate reader/ Microwells:
96 test wells containing anti-Rotavirus antibodies

2. Pipettes

3. Washing system

4. Reagent
• Coated plate
The 96-well plates are made of polystyrene and are coated
with either inactivated antigen or antibody
• Control
to normalize or standardize each plate
• Conjugate
enzyme labeled antibodies that react specifically to plate
bound sample analytes
• Wash concentration
to wash unbound material from the plate.
• Stop solution
stops the enzyme substrate reaction and color development
TYPE OF ELISA TEST
1. Direct ELISA

attachment of an antigen to a polystyrene plate followed by an enzyme-labeled antibody that can react with the antigen and a substrate that
can be measured
Advantages   
Fast and minimal steps involved in the procedure.
Minimum precursor requirement makes it less error prone.

Disadvantages
The immobilization of the antigen is not specific due to which background related issues are seen.
Offers less flexibility in terms of primary antibody.
The absence of signal amplification reduces sensitivity.
TYPE OF ELISA TEST
2. Indirect ELISA

attachment of an antigen to a polystyrene plate followed by an unlabeled or primary antibody followed by an enzyme-labeled antibody that
can react with both the primary antibody and substrate

Advantages
Offers high sensitivity and flexibility as the number of secondary antibodies can bind to a primary antibody and one type of secondary
antibody can label different primary antibodies
It is cheaper as there is a requirement of fewer labeled antibodies.

Disadvantages
Higher signal to noise ratio.
Time consuming and extra labour required.
TYPE OF ELISA TEST
3. Sandwich ELISA

A capture antibody is attached to the polystyrene plate, then antigen is added that specifically attaches or captures the antigen. This second
antibody is then followed by an enzyme-labeled antibody specific for the second antibody that can react with a substrate that can be measured
Advantages   
Offers high sensitivity compared to direct or indirect ELISA
Introduces highly specific reaction due to the involvement of two antibodies for antigen detection.
Both direct and indirect technique is implemented in detection.

Disadvantages
Optimization in terms of antibody becomes problematic due to cross-reactivity issues.
For recognition of a specific epitope, only monoclonal antibodies can be applied as matched pairs.
To procure monoclonal antibodies it is a tedious process in case of matched pairs and are more expensive than polyclonal antibodies.
 TYPE OF ELISA TEST
4. Competitive ELISA
 This test is like the sandwich ELISA but
involves the addition of competing antibodies or
proteins when the second antibody is added.
This results in a decrease in the substrate signal
generated.

Advantage :
High Specificity
Since two antibodies used the antigen/analyte
is specifically captured and detected
Suitable for Complex Samples
Since the antigen does not require purification
prior to measurement
Flexibility and Sensitivity
ROTAVIRUS ELISA TEST
The Rotavirus Stool Antigen ELISA kit is an in vitro assay
for the qualitative determination of rotavirus antigen in feces.
It is a double antibody (sandwich) ELISA using a polyclonal
anti-rotavirus antibody to capture the antigen from the stool
supernatant.
PRINCIPLE ROTAVIRUS STOOL ELISA TEST
During the first incubation, Rotavirus antigens present in the stool supernatant are
captured by antibodies attached to the wells.
The second incubation adds an additional anti-Rotavirus antibody that sandwiches
the antigen.
The third incubation attaches horseradish peroxidase to the sandwich.
After washings to remove unbound enzyme, a chromogen is added which develops a
blue color in the presence of the enzyme complex and peroxide.
The stop solution ends the reaction and turns the blue color to yellow.
PROCEDURE SANDWICH ELISA
Prepare a surface to which a known quantity of antibody is bound.
Add the antigen-containing sample to the plate and incubate the plate at 37°c.
Wash the plate, so that unbound antigen is removed.
Add the enzyme-linked antibodies which are also specific to the antigen and
then incubate at 37°c.
Wash the plate, so that unbound enzyme-linked antibodies are removed.
Add substrate which is converted by the enzyme to produce a colored
product.
Reaction of a substrate with the enzyme to produce a colored product.
Measure the absorbency or fluorescence or electrochemical signal (e.g.,
current) of the plate wells to determine the presence and quantity of antigen