ELISA (Enzyme-Linked Immunosorbent Assay) test


ELISA (Enzyme-Linked Immunosorbent Assay)

1.  Objective

The objective of ELISA was to detect and quantify specific antigens or antibodies in a sample using an enzyme-labeled immunoassay, commonly applied in infectious diseases, hormone testing, and autoimmune diagnostics.

2.  Principle

ELISA works on the principle of antigen-antibody specificity.
An enzyme-labeled antibody or antigen binds to its counterpart in the sample. A substrate is then added, which the enzyme converts into a color product, and the intensity of the color is measured spectrophotometrically, indicating the concentration of the analyte.

Types of ELISA:

  • Direct ELISA – antigen detected by enzyme-linked antibody

  • Indirect ELISA – antigen detected by primary + enzyme-linked secondary antibody

  • Sandwich ELISA – antigen “sandwiched” between two antibodies

  • Competitive ELISA – signal decreases with more antigen in sample

3.  Materials

  • ELISA plate (96-well)

  • Antigen/antibody coated wells

  • Patient serum sample

  • Enzyme-linked antibody (e.g., HRP-conjugated)

  • Substrate (e.g., TMB – tetramethylbenzidine)

  • Stop solution (e.g., H₂SO₄)

  • Microplate reader (at 450 nm)

  • Micropipettes and sterile tips

  • Wash buffer and blocking buffer

4.  Procedure (Sandwich ELISA Example)

  1. Coating: Capture antibody coated onto wells

  2. Blocking: Non-specific sites blocked

  3. Sample addition: Patient’s sample (containing antigen) added

  4. Detection antibody: Enzyme-conjugated antibody added

  5. Washing: Unbound materials washed away

  6. Substrate added: Enzyme converts substrate to color

  7. Stop solution added to stabilize the color

  8. Reading: Optical density (OD) read at 450 nm

5.  Result Interpretation

Sample OD Reading Result
Positive Ctrl 1.650 Valid positive
Negative Ctrl 0.100 Valid negative
Patient A 1.430 Positive
Patient B 0.180 Negative

A cut-off value is usually calculated based on:

Cutoff=Mean Negative Control OD+(2×Standard Deviation)

6.  Example Calculation

Given:

  • Mean Negative Control OD = 0.100

  • SD = 0.030

Cutoff=0.100+(2×0.030)=0.160\text{Cutoff} = 0.100 + (2 × 0.030) = 0.160

If Patient OD = 1.430 → Positive
If Patient OD = 0.180 → Borderline
If Patient OD = 0.140 → Negative

7. Uses of ELISA

  • HIV, Hepatitis B/C detection

  • COVID-19 antibody screening

  • Hormone level testing (e.g., TSH, insulin)

  • Detecting autoimmune antibodies (e.g., ANA, RF)

8.  Conclusion

ELISA was a sensitive and specific assay for detecting a wide range of biomolecules. It was used extensively in clinical diagnostics, research, and vaccine response assessment.


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