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ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a biochemical technique for detecting and quantifying proteins, antibodies, and hormones through antigen-antibody interactions, with various types including direct, indirect, sandwich, and competitive ELISA. The procedure involves coating, blocking, binding, detection, substrate addition, and measurement, making it widely applicable in medical diagnostics, forensic science, and biotechnology. While ELISA is sensitive and cost-effective, it requires specific antibodies and can produce false results due to cross-reactivity.

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41 views3 pages

ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a biochemical technique for detecting and quantifying proteins, antibodies, and hormones through antigen-antibody interactions, with various types including direct, indirect, sandwich, and competitive ELISA. The procedure involves coating, blocking, binding, detection, substrate addition, and measurement, making it widely applicable in medical diagnostics, forensic science, and biotechnology. While ELISA is sensitive and cost-effective, it requires specific antibodies and can produce false results due to cross-reactivity.

Uploaded by

Hamid Anees
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

Enzyme-Linked Immunosorbent Assay (ELISA)

Definition:
ELISA (Enzyme-Linked Immunosorbent Assay) is a biochemical technique used to detect and
quantify substances such as proteins, peptides, antibodies, and hormones. It is widely used in
diagnostics, forensic science, biotechnology, and research to identify specific antigens or
antibodies in biological samples.

Principle of ELISA
ELISA is based on the antigen-antibody interaction. An enzyme is linked to an antibody or
antigen, and when a substrate is added, the enzyme catalyzes a color change, which is measured
spectrophotometrically. The intensity of the color change is proportional to the concentration of
the target molecule in the sample.

Types of ELISA
1. Direct ELISA
o A primary antibody directly conjugated with an enzyme binds to
the target antigen.
o The substrate is added to produce a detectable signal.
o Advantage: Simple and quick.
o Disadvantage: Less sensitivity and specificity.

2. Indirect ELISA
o A primary antibody binds to the target antigen, and a secondary
enzyme-linked antibody binds to the primary antibody.
o Advantage: Higher sensitivity due to signal amplification.
o Disadvantage: More steps, leading to possible cross-reactivity.

3. Sandwich ELISA
o Uses two antibodies: a capture antibody coated on a plate and a
detection antibody.
o The antigen binds between these two antibodies.
o Advantage: Highly specific and sensitive.
o Disadvantage: Requires matched antibody pairs, making it
complex.

4. Competitive ELISA
o The sample antigen competes with a labeled antigen for binding
sites on an antibody.
o Advantage: Suitable for detecting small molecules and toxins.
o Disadvantage: More complex analysis.

Procedure of ELISA
1. Coating: The antigen or antibody is immobilized onto a microplate
well.
2. Blocking: A blocking agent (e.g., bovine serum albumin) prevents
non-specific binding.
3. Binding: The sample containing the target antigen or antibody is
added.
4. Detection: An enzyme-linked secondary antibody is added to bind the
target molecule.
5. Substrate Addition: A chromogenic substrate (e.g., TMB) is added,
producing a color change.
6. Measurement: The intensity of color is measured using a
spectrophotometer at a specific wavelength.

Applications of ELISA
 Medical Diagnostics:
o Detection of diseases like HIV, COVID-19, hepatitis, and
tuberculosis.
o Pregnancy tests.
 Forensic Science:
o Identification of blood proteins and drugs in biological samples.
 Food Industry:
o Detection of allergens (e.g., gluten, peanut proteins).
 Biotechnology & Research:
o Measurement of cytokines, hormones, and growth factors.

Advantages of ELISA
 High sensitivity and specificity.
 Can detect even small concentrations of analytes.
 Suitable for large-scale screening.
 Relatively simple and cost-effective compared to other immunoassays.
Limitations of ELISA
 Requires specific antibodies for detection.
 False positives or negatives can occur due to cross-reactivity.
 Some forms of ELISA require expensive equipment.

Conclusion

ELISA is a crucial tool in diagnostics, forensic science, and biotechnology, offering a highly
sensitive and specific method for detecting biomolecules. Its versatility makes it an essential
technique in clinical and research laboratories worldwide.

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