College of health and medical technology / Baghdad
Department of medical laboratory techniques
Enzyme-Linked Immunosorbent Assay
(ELISA)
By: Qudamah Jasim Dawood
Enzyme-Linked Immunosorbent Assay (ELISA)
Introduction:
ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique involving
the reaction of antigen and antibody in well. It is a highly sensitive and specific method
for the detection and quantification of antigens or antibodies. ELISA tests are usually
performed in micro-well plates.
Principal:
The Enzyme-Linked Immunosorbent Assay (ELISA) is based on the specific binding
between an antigen and its corresponding antibody. In this method, either the antigen or
antibody is fixed onto a solid surface, usually a micro plate well. The test sample is then
added, allowing the target molecule to bind specifically. Detection is achieved by using
an enzyme-conjugated antibody, which reacts with a suitable substrate to produce a
measurable color change. The intensity of the color is directly related to the concentration
of the target antigen or antibody in the sample.
Component of ELISA:
• Coated Plates
The 96-well plates are made of polystyrene coated with either
inactivated antigen or antibody. This coating is the binding site for the
antibodies or antigens in the sample.
• Sample Diluent
Most assays require a specific dilution of the sample. Samples are
added to the sample diluent and mixed prior to putting them onto the
coated plates.
• Controls
The positive control is a solution that contains antibody or antigen.
The negative control is a solution without antibody or antigen.
• Conjugate
ELISA conjugates are enzyme-labeled antibodies or antigens that
react specifically to plate-bound sample analytes.
• Standards
For calibration curve construction, thought quantitative measurement.
• Substrate
For peroxidase conjugates, the substrate is a mixture of hydrogen peroxide
and a chromogen that reacts with the enzyme portion of the conjugate to
produce color.
• Wash Concentrate
The wash concentrate is a buffered solution containing detergent used to
wash away unbound materials from the plates.
• Stop Solution
The stop solution is a strong acid or base that stops the enzyme–substrate
reaction and, thereby, the color development.
Types of ELISA
• Indirect ELISA:
Indirect ELISA is a two-step binding process involving the use of a primary
antibody and a labeled secondary antibody. In this method, the primary
antibody is incubated with the antigen-coated wells.Next, a labeled
secondary antibody that recognizes the primary antibody is added. A
substrate is then added to the well to produce a signal amplification.
Features: High sensitivity,The indirect ELISA is most suitable for
determining total antibody concentration in samples.
• Sandwich ELISA:
In sandwich ELISA, the plate is coated with a capture antibody, which
binds to the target antigen. A second enzyme-linked antibody is added
to form an antigen-antibody sandwich.
Features: Very speci ic, highly sensitive, and Suitable for detecting very
low concentrations of proteins
f
• Competitive ELISA :
In this assay, the antigen in the sample competes with an enzyme-labeled
antigen for limited antibody binding sites. When the sample has a high
antigen concentration, more unlabeled antigen binds, and less labeled
antigen is attached.After adding the chromogenic substrate, samples with
high antigen levels produce a weaker color signal, while those with low
antigen levels give a stronger signal. there is an inverse relationship between
antigen concentration and color intensity.
Applications of ELISA:
ELISA is applied in various scientific and clinical fields, including:
• Detect and Estimate Hormone Levels:
TSH , HCG , prolactin
• Screening Donated Blood for Possible Viral Contaminants:
-HIV1/2 Ag-Ab 4th generation , syphilis (TP)
-HCV - Ab , HBS -Ag , HBC - total Ab
• Detect and Estimate tumor marker Levels:
• Drug testing and detection of toxins.
