ELISA (enzyme-linked immunosorbent assay) is a highly sensitive technique used to detect specific proteins like antigens or antibodies in samples. It involves an antigen-antibody reaction, an enzymatic reaction producing a colored product, and detection of the color. There are different types of ELISA including direct, indirect, sandwich, and competition assays. ELISA provides quantitative and qualitative results by measuring color change from an enzymatic substrate reaction, allowing determination of protein presence and concentration in samples through comparison to a standard curve.

1. ELISA
Enzyme-Linked Immunosorbent Assay
Hafsa Ijaz, R.Ph, M.Phil Pharmacology Scholar, 2022
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2. ELISA
Enzyme Linked Immunosorbent Assay
(ELISA) is highly sensitive immunochemical
technique which is used to access the presence of
specific protein (antigen or antibody) in the given
sample and it’s quantification.
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3. Principle
• Plate-based assay technique. Along with the enzyme-labeling
of antigens or antibodies, it involves three principles in
combination which make it one of the most specific and sensitive
than other immunoassays to detect the biological molecule:
o An immune reaction i.e. antigen-antibody reaction.
o Enzymatic chemical reaction i.e. enzyme catalyzes the
formation of colored (chromogenic) product from colorless
substrate.
o Signal detection and Quantification i.e. detection and
measurement of color intensity of the colored products generated
by the enzyme and added substrate.
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4. ELISA Plates
• Flat-bottomed 96 well
plates
• Made from polystyrene or
polyvinyl chloride
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5. Antigen
Enzyme
(HRP)
Antibody
Enzyme conjugated
detection antibody
*Several washed repeated to remove
access antigen.
*Access liquid is removed to prevent
dilution of solution added in next step.
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6. HRP:
• horseradish peroxidase (enzyme found in roots of
horseradish a root vegetable of family Brassicaceae) is
glycoprotein in nature produces color when incubated
with proper substrate).
TMB Core +:
• TMB Core+ is used a substrate for HRP.
• It contains; TMB(3,3,5,5-tetramethyl benzamide)
solution, buffer and hydrogen peroxide.
• Produces deep blue color, read at 655nm.
• Reaction stopped with sulfuric acid, resulting in
yellow color, read at 450nm.
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7. Coating buffer:
• Used to coat ELISA multiwell plate
• Maximize adsorption to the plate
• Optimize interaction with detection antibody
• Example; bicarbonate buffer, pH 9.6
Blocking buffer:
• Used to prevent nonspecific binding of detection
antibodies to multiwell plate surface.
• They are of 2 types; proteins(blocks permanently) and
detergents(blocks temporarily-disappears after washing).
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8. Types of ELISA
1.Direct ELISA
2.Indirect ELISA
3.Sandwich ELISA
4.Competition/Inhibition ELISA
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9. Direct ELISA
1. Antigen is
immobilized in the well
of ELISA plate.
2. Antigen is detected by
antibody-conjugated with
enzyme(HRP).
3. Substrate is catalyzed
by enzyme to generate
colored readout (substrate
solution’s color is
changed).
Best for: When analyzing immune response for an antigen
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10. Indirect ELISA
1. Antigen is
immobilized in
the well of ELISA
plate.
2. Unlabeled primary
antibody binds to specific
antigen.
4. Substrate is
catalyzed by
enzyme to generate
colored readout
(substrate solution’s
color is changed).
3. Enzyme conjugated
secondary antibody,
directed against host
species of primary
antibody.
Best for: Determining total antibody concentration in a sample
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11. Sandwich ELISA
1. Well of ELISA
plate, coated with
capture antibody.
2. Analyte or sample of
antigen is added.
4. Substrate is
catalyzed by enzyme
to generate colored
readout (substrate
solution’s color is
changed).
3. Enzyme conjugated
detection antibody is
added.
*2-5 times more sensitive than direct or indirect ELISA
Best for: Analysis of complex samples, since the antigen does not need to be purified prior to
measurement.
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12. Competition/
Inhibition
ELISA
Antigens
Detection
Antibody
Enzyme conjugated
Secondary detection
antibody
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13. • Most of the Antigens occupied by
enzyme-linked antibodies.
• More absorbance (because enzyme
conjugated with secondary antibody
generated color with substrate).
• Concentration of antibodies in sample
is less.
• Most of the antigens are
occupied by antibodies from
sample.
• Less absorbance.
• Concentration of antibodies in
sample is more.
Detection
Antibody
Enzyme conjugated
Secondary detection
antibody
Best for: When only one antibody is available for antigen of interest. Detection of small
antigen that cannot be bound by two different antibodies such as in Sandwich ELISA.
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14. Results
Detection: HRP and substrate produces colorimetric output that
is read by spectrophotometer.
Quantitative:
• Data is interpreted in comparison to standard curve
• Precisely calculate concentration of antigen in various sample.
Qualitative:
• Gives yes or no indication for presence or absence of antigen.
• Sample is compared to a blank well, containing no antigen or
unrelated control antigen
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15. Reference
 The Immunoassay Hand Book by, David Wild, 2013.
 Application of ELISA by, Willem Duermeyer 1990.
 ELISA Basic Guide by, Bio-Rad Laboratories, 2020.
 Evaluation of ELISA for Antigen and Antibody Detection by,
Miriam Valesco, 1986.
 ELISA Techniques by, Gillian R. Bullock, ‎
Daniel van Velzen, ‎
M.
J. Warhol, 1991
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