Primary Binding Assays
Primary binding assays directly measure the binding of antibodies and their specific antigens. These assays focus on detecting and quantifying the direct interaction between antigens and antibodies. In this chapter we will discuss some of the most commonly used primary binding assays:
Enzyme-Linked Immunosorbent Assay (ELISA)
Immunochromatographic Assay (ICA)
Radioimmunoassay (RIA)
A) Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme-linked immunosorbent Assay (ELISA) is a widely used immunological technique designed for the detection and quantification of specific proteins, typically antigens or antibodies, in biological samples.
Principle of ELISA:
The principle behind ELISA is the antigen-antibody interaction. Antibodies can bind specifically to their corresponding antigen, forming an antigen-antibody complex. ELISA takes advantage of this specificity by using enzymes linked to either the antigen or antibody, which react with a substrate to produce a detectable signal, such as a colour change.
There are several types of ELISA, each designed for specific purposes. The main types of ELISA include:
Figure: Types of ELISA- Direct, Indirect, Sandwich, and Competitive
Direct ELISA: In a direct ELISA, the antigen is immobilized directly onto the microplate surface. The primary antibody, which is specific to the antigen of interest, is then added directly to the well. A secondary antibody labelled with an enzyme is used to detect the binding of the primary antibody.
Indirect ELISA: In an indirect ELISA, the antigen is immobilized onto the microplate surface, similar to the direct method. Instead of adding the primary antibody directly, a primary antibody specific to the antigen is added, followed by a secondary antibody labelled with an enzyme.
Sandwich ELISA: In a sandwich ELISA, the antigen is trapped between two layers of antibodies- a capture antibody and a detection antibody. The capture antibody is immobilized on the microplate and binds to the antigen. The detection antibody, labelled with an enzyme, is then added to detect the bound antigen.
Competitive ELISA: In competitive ELISA, a labelled antigen competes with the sample antigen for binding to a limited amount of immobilized antibodies. The amount of labelled antigen bound is inversely proportional to the concentration of the antigen in the sample.
B) Immunochromatographic Assay (ICA)
Immunochromatographic Assay (ICA), also known as lateral flow or rapid tests, is a technique that enables the quick and simple detection of specific target molecules, such as antigens or antibodies, in biological samples. A common example of immunochromatographic assay includes the RSID tests for the detection of human blood, semen, saliva, urine, etc.
Principle of Immunochromatographic Assay (ICA):
Immunochromatographic assays are based on the principles of capillary action and specific antigen-antibody interactions to enable rapid and visual detection of target analytes. This rapid test utilizes a test strip containing distinct zones, including a sample application pad, a conjugate pad containing labelled primary antibodies, a reaction or test zone with immobilized primary antibodies, and a control zone with immobilized secondary antibodies.
Figure: Principle of Immunochromatographic Assay (ICA)
When a sample is added to the application pad it begins to migrate along the membrane facilitated by capillary action. The target antigens, if present in the sample, form an antigen-antibody complex with labelled primary antibodies on the conjugate pad. These complexes are captured at the test zone (T) and the control zone (C), resulting in the formation of coloured lines at these zones. The appearance or absence of these lines indicates the test result.
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C) Radioimmunoassay (RIA)
Radioimmunoassay (RIA) is a powerful analytical technique used for the quantification of specific molecules, including antigens, antibodies, hormones, and other analytes present in biological samples. Unlike conventional assays, RIA utilizes the unique properties of radioactive isotopes to trace and measure the concentration of target molecules.
Figure: Principle of Radioimmunoassay (RIA)
Principle of Radioimmunoassay (RIA):
Radioimmunoassay is based on the competitive binding of a radioactive-labelled antigen (tracer) and the unlabelled antigen in the test sample to a limited amount of antibody. The amount of detected radioactivity of the bound tracer inversely correlates with the concentration of the target antigen in the sample.