🔅ELISA is a technique that uses antibodies and enzymes to detect and measure antigens in a sample.

BASIC STEPS OF ELISA:

• First, the antigen is immobilized on a solid surface, such as a microtiter plate. This can be done by directly coating the antigen on the plate, or by using a capture antibody that binds to the antigen and attaches it to the plate.

• Second, the detection antibody is added to the plate. This antibody is specific for the antigen and binds to it, forming a complex. The detection antibody is also linked to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which can catalyze a chemical reaction that produces a color or light signal.

• Third, any unbound detection antibodies are washed away from the plate, leaving only the antigen-antibody-enzyme complex on the surface.

• Fourth, a substrate is added to the plate. This substrate is a molecule that reacts with the enzyme and produces a detectable signal. For example, HRP can oxidize TMB (3,3',5,5'-tetramethylbenzidine) and produce a blue color, while AP can hydrolyze pNPP (p-nitrophenyl phosphate) and produce a yellow color. The intensity of the signal is proportional to the amount of antigen in the sample.

• Fifth, the signal is measured by a spectrophotometer or a luminometer, depending on the type of substrate used. The signal can be compared to a standard curve or a control sample to calculate the concentration or presence of the antigen in the sample.

TYPES OF ELISA TESTS

– There are different types of ELISA, depending on how the antigen and the detection antibody are applied to the plate. Some of the common types of ELISA are:

🔅 Direct ELISA

– For this test, the antigen is directly coated on the plate, and then detected by an enzyme-linked antibody. This type of ELISA is simple and fast, but it may have low specificity and high background noise due to non-specific binding of antibodies.

🔅 Indirect ELISA

For this test, the antigen is directly coated on the plate, and then detected by an unlabeled primary antibody followed by an enzyme-linked secondary antibody. This type of ELISA is more sensitive and specific than direct ELISA, as it allows for signal amplification and reduces cross-reactivity.

🔅 Sandwich ELISA

– For this test, the antigen is captured by an antibody coated on the plate, and then detected by another enzyme-linked antibody. This type of ELISA is highly sensitive and specific for large antigens with multiple epitopes, but it requires two different antibodies that recognize different regions of the antigen.

🔅 Competitive ELISA

– For this test, the antigen in the sample competes with a known amount of enzyme-linked antigen for binding to an antibody coated on the plate. The signal is inversely proportional to the amount of antigen in the sample. This type of ELISA is useful for small antigens with a single epitope, but it requires careful optimization of the assay conditions.