Immunoassay techniques are analytical methods that utilize the specific binding interaction between an antibody and an antigen to detect and quantify the presence of a target substance, such as a protein or chemical compound.

Get the full solved assignment PDF of MEVE-018 of 2023-24 session now.

These techniques are widely used in medical diagnostics, environmental monitoring, and various research applications. One popular type of immunoassay is the enzyme immunoassay (EIA), also known as enzyme-linked immunosorbent assay (ELISA). Here’s an explanation of how enzyme immunoassays work:

Working of Enzyme Immunoassays (ELISA):

Basic Components:

Coating:

A solid surface, such as a microplate well, is coated with an antigen or antibody. This immobilized component is used to capture the target analyte.

Blocking:

To prevent nonspecific binding, the coated surface is typically blocked with a blocking agent (e.g., bovine serum albumin) that covers unoccupied binding sites.

Sample Application:

The sample containing the target analyte is added to the coated surface. If the target is present, it will bind specifically to the immobilized antigen or antibody.

Washing:

Unbound substances are removed by washing the solid phase to reduce background noise and enhance specificity.

Addition of Detection Antibody:

A labeled antibody (detection antibody) that is specific to a different epitope on the target analyte is added. This antibody is conjugated to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).

Washing (again):

Excess detection antibody is washed away, leaving only the specifically bound enzyme-labeled antibody on the solid surface.

Enzyme Substrate Addition:

A substrate solution containing a color-changing or fluorescent substrate for the enzyme is added. The enzyme catalyzes a reaction that produces a detectable signal.

Signal Detection:

The intensity of the signal is directly proportional to the amount of bound enzyme-labeled antibody, which, in turn, is proportional to the amount of target analyte in the sample.

Measurement:

The color change or fluorescence is measured using a spectrophotometer or a fluorescence reader. The results are typically quantified by comparing the signal from the sample to a standard curve generated from known concentrations of the target analyte.

Types of Enzyme Immunoassays:

Direct ELISA:

The antigen is directly immobilized on the solid surface, and a labeled antibody specific to the antigen is used for detection.

Indirect ELISA:

The immobilized antigen captures the target, and a secondary antibody labeled with an enzyme is used for detection. This secondary antibody recognizes the primary antibody bound to the target.

Sandwich ELISA:

The target analyte is captured between two antibodies – one immobilized on the solid surface and the other labeled with an enzyme. This format is highly specific and is often used for detecting antigens.

Competitive ELISA:

A labeled antigen competes with the sample antigen for binding to a limited number of immobilized antibodies. The amount of signal inversely correlates with the concentration of the target analyte in the sample.

Enzyme immunoassays offer high sensitivity, specificity, and ease of use. They are employed in various fields, including clinical diagnostics for detecting diseases, monitoring hormone levels, detecting allergens, and assessing the presence of environmental contaminants or infectious agents.