ELISA Protocol: Principle, Steps, and Applications

Introduction to ELISA

The Enzyme-Linked Immunosorbent Assay (ELISA) is a highly sensitive immunological technique used to detect and quantify proteins, antibodies, antigens, and hormones. It is widely applied in clinical diagnostics, biotechnology, food safety, and research laboratories due to its accuracy, specificity, and adaptability.

Principle of ELISA

ELISA is based on the specific antigen-antibody interaction. The antigen (or antibody) of interest is immobilized on a solid surface (usually a 96-well microplate), and detection is achieved through an enzyme-conjugated antibody. When a suitable substrate is added, the enzyme catalyzes a reaction that produces a measurable color change, proportional to the amount of analyte present.Read more

Types of ELISA

1. Direct ELISA:  Antigen immobilized, detected by an enzyme-linked primary antibody.
2. Indirect ELISA : Antigen immobilized, detected by a primary antibody and a labeled secondary antibody.
3. Sandwich ELISA : Uses a "capture" antibody to bind the antigen, followed by detection with a second antibody.
4. Competitive ELISA : Antigen in the sample competes with a labeled antigen for binding to the capture antibody.

Materials Needed

• 96-well microtiter plate (polystyrene)
• Coating buffer (carbonate-bicarbonate buffer, pH 9.6)
• Washing buffer (PBS or TBS with 0.05% Tween-20)
• Blocking buffer (BSA, skimmed milk, or casein)
• Primary antibody / Antigen
• Enzyme-conjugated secondary antibody (commonly HRP or AP)
• Substrate (TMB for HRP, PNPP for AP)
• Stop solution (H₂SO₄ for HRP-based ELISA)
• Microplate reader (450 nm for TMB substrate)

Step-by-Step ELISA Protocol (Indirect ELISA Example)

1. Coating the Plate

• Add 100 µL of antigen diluted in coating buffer to each well.
• Incubate overnight at 4°C or 2 hours at 37°C.
• Wash 3 times with washing buffer to remove unbound antigen.

2. Blocking

• Add 200 µL of blocking buffer (1% BSA in PBS) to each well.
• Incubate for 1–2 hours at room temperature.
• Wash 3 times to remove excess blocking solution.

3. Primary Antibody Incubation

• Add 100 µL of primary antibody diluted in PBS-Tween.
• Incubate for 1–2 hours at room temperature or overnight at 4°C.
• Wash 3–5 times.

4. Secondary Antibody Incubation

• Add 100 µL of enzyme-conjugated secondary antibody.
• Incubate for 1 hour at room temperature.
• Wash 5 times to reduce background noise.

5. Substrate Addition

• Add 100 µL of substrate solution (TMB for HRP).
• Incubate in the dark for 10–30 minutes until color develops.

6. Stop Reaction

• Add 50 µL of stop solution (1N H₂SO₄ for HRP assays).
• Color changes from blue to yellow (in TMB).

7. Measurement

• Read absorbance using a microplate reader at 450 nm.
• Compare optical density (OD) values to a standard curve for quantification.

Data Analysis

• Create a standard curve using known concentrations of antigen.
• Plot absorbance (OD) vs. concentration.
• Determine unknown sample concentrations by interpolation.
• Positive samples show higher absorbance compared to negative controls.

Applications of ELISA

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• Clinical Diagnostics: Allergies, hormone detection (hCG, insulin).
• Research: Protein quantification, cytokine analysis, antibody titer measurement.
• Food Industry: Detection of allergens (gluten, peanuts) and pathogens.
• Pharmaceutical Industry: Drug monitoring, vaccine development.

Advantages

• High sensitivity and specificity.
• Suitable for large-scale screening.
• Quantitative and qualitative results.
• Cost-effective compared to other immunoassays.

Limitations

• Requires well-optimized conditions.
• Risk of cross-reactivity and false positives.
• Multiple washing steps increase assay time.