Indirect ELISA

The steps of "indirect" ELISA follows the mechanism below:-

·         A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.

·         A solution of non-reacting protein, such as bovine serum albumin or casein, is added to block any plastic surface in the well that remains uncoated by the antigen.

·         Next the primary antibody is added, which binds specifically to the test antigen that is coating the well. This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen.

·         Afterwards, a secondary antibody is added, which will bind the primary antibody. This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody.

·         A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. This can be helpful in a clinical setting, and in R&D.

·         The higher the concentration of the primary antibody that was present in the serum, the stronger the color change. Often a spectrometer is used to give quantitative values for color strength.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. Within common-sense limitations, the enzyme can go on producing color indefinitely, but the more primary antibody is present in the donor serum the more secondary antibody + enzyme will bind, and the faster color will develop. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; when serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich or direct ELISA provides a solution to this problem, by using a "capture" antibody specific for the test antigen to pull it out of the serum's molecular mixture.

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation (error inherent in a test) is often used to distinguish positive from negative samples. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule. For example, if a test sample returns an OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same analyte concentration as your sample.