ELISA (Enzyme-linked Immunosorbent Assay)


HIV ELISA

An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in an 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which is about 1 cm high and 0.7 cm in diameter.

When the body is infected with a virus, the immune system responds by producing antibodies–proteins that circulate in the blood and attempt to destroy the virus. If antibodies against HIV are present in a blood sample, they will stick to the plate coated with fragments of the virus. The ELISA reagent will detect the presence of the bound antibodies and will change colour giving a positive result.

Partially purified, inactivated HIV antigens are coated onto an ELISA plate. Patient’s blood serum which contains antibodies to HIV is added and those antibodies will bind to the HIV antigens on the plate. Anti-human immunoglobulin is coupled to an enzyme. This is the second antibody that binds to human antibodies. Chromogen or substrate which changes colour when cleaved by the enzyme is attached to the second antibody.

In most cases if the ELISA tests are positive, the patient will be retested by the western blotting analysis again for confirmation.

TYPES OF ELISA

Generally there are 5 types of ELISA: Direct ELISA; Indirect ELISA; Sandwich ELISA; Competitive ELISA and Multiplex ELISA. The two common types of ELISA are indirect ELISA (that detect antibodies in sample) and sandwich ELISA (that detect antigen in sample).

DIRECT ELISA

The direct ELISA uses the method of directly labelling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labelled antibody is quantitated by a colorimetric, chemiluminescent or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample.

INDIRECT ELISA

The indirect ELISA is a two-step method that uses a labelled secondary antibody for detection. Apply a sample of known antigen of known concentration to the well of a microtiter plate. A concentrated solution of non-interacting protein, such as bovine serum albumin or casein, is added to all plate wells for blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.

The plate wells are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.

Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.

View the result using a spectrophotometer, spectrofluorometer, or other optical or electrochemical devices.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules.

SANDWICH ELISA

The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to the antibodies, since at least two antibodies act in the sandwich. To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash and a labelled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”.

Prepare a surface to which a known quantity of capture antibody is bound. Block any non specific binding sites on the surface by adding serum proteins. Apply the antigen-containing sample to the plate. Wash the plate, so that unbound antigen is removed. Apply primary antibodies that bind specifically to the antigen. Apply enzyme-linked secondary antibodies which are specific to the primary antibodies. Wash the plate, so that the unbound antibody-enzyme conjugates are removed. Apply a chemical which is converted by the enzyme into a colour or fluorescent or electrochemical signal. Measure the absorbance or fluorescence or electrochemical signal of the plate wells to determine the presence and quantity of antigen.

COMPETITIVE ELISA

When two “matched pair” antibodies are not available for a target, another option is the competitive ELISA. The advantage of competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the antigen or the primary antibody. In this type of ELISA, there is an inverse relationship between the signal obtained and the concentration of the analyte in the sample, due to the competition between the free analyte and the ligand-enzyme conjugate for the antibody coating the microplate, i.e. the more analyte the lower the signal.

An unlabelled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards. After this reaction is allowed to go to equilibrium, conjugated antigen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabelled antigen. Thus, the more unlabelled antigens in the sample or standard, the lower the amount of conjugated antigen bound. The plate is then developed with substrate and colour change is measured.

MULTIPLEX ELISA

 In this ELISA a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Antigens are measured by coating or printing capture antibodies in an array format within a single well to allow for the construction of “sandwich” ELISA quantification assays. Generally, multiplex ELISA can also be achieved through antibody array, where different primary antibodies can be printed onto the glass plate to capture corresponding antigens in a biological sample such as plasma, cell lysate, or tissue extracts. Detection method can be direct or indirect, sandwich or competitive, labelling or non-labelling, depending upon antibody array technologies.

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