ELISA or enzyme-linked immunosorbent assay is a diagnostic method used in detecting the presence of antibody or an antigen in a particular sample. This biochemical technique has been very useful in medicine particularly in immunology and in plant pathology, and has divided into four different types: Direct and Indirect ELISA, Sandwich, Competitive, and Multiplex ELISA.
· Direct ELISA is a method of directly labeling the antibody itself. The microwell plates which are coated with a sample containing the antigen and the labeled antibody are quantitated using a chemiluminiscent, colorimetric or fluorescent end-point. This method avoids the secondary antibody to cross-react with the antigen sample components. However, direct ELISA consumes more time to carry out and requires expensive proposition.
· Indirect ELISA is a two-step method that uses a primary and secondary labeled antibody. The primary antibody is incubated with the antigen followed by the incubation of the secondary antibody. But, this may give a nonspecific signal result because cross-reaction with the secondary antibody may occur.
· Sandwich ELISA measures the amount of antigens between a two given antibody layers. The antigens contain at least two antigenic spots since at least two antibodies act in the sandwich. In performing this assay, the first antibody is purified and bound to a solid phase attached to the bottom of the plate. Then an antigen is added and allowed to combine with the bound antibody while the unbound products are removed. Finally, the second antibody is bound to the antigen completing the sandwich ELISA.
· Competitive ELISA is an assay that does not require a “matched pair” antibodies. In this method, one reagent must be paired to a detection enzyme (horseradish or peroxidase) and then the enzyme is linked to another antibody. With competitive ELISA an opposite relationship between the signal and the concentration of the analyte in the sample is obtained.
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