Here are the main types of ELISA:

1. Direct ELISA

Procedure:

• The antigen is immobilized directly onto the microplate.
• A primary antibody conjugated to an enzyme is added, which binds to the antigen.
• After washing to remove unbound antibodies, a substrate is added that reacts with the enzyme to produce a measurable signal.

Advantages:

• Simple and quick, with fewer steps and reagents.
• Reduced potential for cross-reactivity since only one antibody is used.

Disadvantages:

• Less flexible since the primary antibody must be labeled with an enzyme.
• Lower signal amplification and sensitivity compared to other ELISA types.

2. Indirect ELISA

Procedure:

• The antigen is immobilized on the microplate.
• An unlabeled primary antibody is added to bind the antigen.
• A labeled secondary antibody (conjugated to an enzyme) that recognizes the primary antibody is added.
• After washing, a substrate is added, and the enzyme reaction produces a detectable signal.

Advantages:

• Increased sensitivity due to signal amplification from the secondary antibody.
• More flexible, as the same labeled secondary antibody can be used with different primary antibodies.

Disadvantages:

• Additional steps and reagents increase complexity and time.
• Potential for cross-reactivity from the secondary antibody.

3. Sandwich ELISA

Procedure:

• A capture antibody is immobilized on the microplate.
• The sample containing the antigen is added, and the antigen binds to the capture antibody.
• A detection antibody (specific to a different epitope on the antigen) is added.
• A secondary antibody conjugated to an enzyme (if the detection antibody is not labeled) is added.
• After washing, a substrate is added, and the enzyme reaction produces a detectable signal.

Advantages:

• High specificity since two different antibodies bind to the antigen.
• High sensitivity due to multiple binding events and signal amplification.

Disadvantages:

• More complex and time-consuming with multiple antibodies and steps.
• Requires well-characterized pairs of antibodies for each target antigen.

4. Competitive ELISA

Procedure:

• The target antigen in the sample and a known amount of enzyme-labeled antigen compete for binding to a limited number of antibody sites.
• The antibody-antigen complexes are then immobilized on the microplate.
• The bound enzyme-labeled antigen produces a signal inversely proportional to the amount of target antigen in the sample.

Advantages:

• Suitable for detecting small antigens that may have only one epitope.
• Can be used for measuring low concentrations of antigen.

Disadvantages:

• Requires precise optimization and controls.
• More complex interpretation of results due to inverse relationship.

5. Multiplex ELISA

Procedure:

• Multiple capture antibodies are immobilized on distinct regions or wells of the microplate.
• The sample is added, and multiple antigens bind to their respective capture antibodies.
• Detection antibodies, specific to each antigen, are added.
• The detection process involves secondary antibodies conjugated to different enzymes or fluorescent labels.
• Signals from each enzyme or label are measured simultaneously.

Advantages:

• Allows simultaneous detection of multiple analytes in a single sample.
• Efficient use of sample volume and reagents.

Disadvantages:

• Requires careful design to avoid cross-reactivity and ensure accurate multiplexing.
• More complex data analysis.

Each type of ELISA has its specific applications and is chosen based on the requirements of sensitivity, specificity, sample type, and the availability of antibodies.