There Are Numerous Variants of ELISA

A number of variations of ELISA have been developed, allowing qualitative detection or quantitative measurement of either antigen or antibody. Each type of ELISA can be used qualitatively to detect the presence of antibody or antigen. Alternatively, a standard curve based on known concentrations of antibody or antigen is prepared, from which the unknown concentration of a sample can be determined.

INDIRECT ELISA

Antibody can be detected or quantitatively determined with an indirect ELISA (Figure 6-10a). Serum or some other sample containing primary antibody (Ab1) is added to an antigen-coated microtiter well and allowed to react with the antigen attached to the well. After any free Ab1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), which binds to the primary antibody. Any free Ab2 then is washed away, and a substrate for the enzyme is added. The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds.

Indirect ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS. In this assay, recombinant envelope and core proteins of HIV are adsorbed

(a) Indirect ELISA

Antigen-coated well wash

Add specific antibody to be measured

Antigen-coated well

Add specific antibody to be measured wash

Add enzyme-conjugated secondary antibody

Add enzyme-conjugated secondary antibody wash

Add substrate (S) and measure color

Add substrate (S) and measure color

(b) Sandwich ELISA

Antibody-coated well wash

Add antigen to be measured

Antibody-coated well

Add antigen to be measured wash

Add enzyme-conjugated secondary antibody wash

Add enzyme-conjugated secondary antibody

Add substrate and measure color

Add substrate and measure color

(c) Competitive ELISA

Incubate antibody with antigen to be measured

Add Ag-Ab mixture to antigen-coated well

FIGURE 6-10

Variations in the enzyme-linked immunosorbent assay (ELISA) technique allow determination of antibody or antigen. Each assay can be used qualitatively, or quantitatively by comparison with standard curves prepared with known concentrations of antibody or antigen. Antibody can be determined with an indirect ELISA

wash v

Add enzyme-conjugated secondary antibody wash t* Ss •A..-

Add substrate and measure color

(a), whereas antigen can be determined with a sandwich ELISA (b) or competitive ELISA (c). In the competitive ELISA, which is an inhibition-type assay, the concentration of antigen is inversely proportional to the color produced.

as solid-phase antigens to microtiter wells. Individuals infected with HIV will produce serum antibodies to epitopes on these viral proteins. Generally, serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of infection.

SANDWICH ELISA

Antigen can be detected or measured by a sandwich ELISA (Figure 6-10b). In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well. A sample containing antigen is added and allowed to react with the immobilized antibody. After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.

COMPETITIVE ELISA

Another variation for measuring amounts of antigen is competitive ELISA (Figure 6-10c). In this technique, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigen-coated microtiter well. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well as in an indirect ELISA. In the competitive assay, however, the higher the concentration of antigen in the original sample, the lower the absorbance.

CHEMILUMINESCENCE

Measurement of light produced by chemiluminescence during certain chemical reactions provides a convenient and highly sensitive alternative to absorbance measurements in ELISA assays. In versions of the ELISA using chemiluminescence, a luxogenic (light-generating) substrate takes the place of the chromogenic substrate in conventional ELISA reactions. For example, oxidation of the compound luminol by H2O2 and the enzyme horseradish peroxidase (HRP) produces light:

luminol + H7O7

light

The advantage of chemiluminescence assays over chro-mogenic ones is enhanced sensitivity. In general, the detection limit can be increased at least ten-fold by switching from a chromogenic to a luxogenic substrate, and with the addition of enhancing agents, more than 200-fold. In fact, under ideal conditions, as little as 5 X 10"18 moles (5 attomoles) of target antigen have been detected.

ELISPOT ASSAY

A modification of the ELISA assay called the ELISPOT assay allows the quantitative determination of the number of cells in a population that are producing antibodies specific for a given antigen or an antigen for which one has a specific antibody (Figure 6-11). In this approach, the plates are coated with the antigen (capture antigen) recognized by the antibody of interest or with the antibody (capture antibody) specific for the antigen whose production is being assayed. A suspension of the cell population under investigation is then added to the coated plates and incubated. The cells settle onto the surface of the plate, and secreted molecules reactive with the capture molecules are bound by the capture molecules in the vicinity of the secreting cells, producing a ring of antigen-antibody complexes around each cell that is producing the molecule of interest. The plate is then washed and an enzyme-linked antibody specific for the secreted antigen or specific for the species (e.g., goat anti-rabbit) of the secreted antibody is added and allowed to bind. Subsequent development of the assay by addition of a suitable chromogenic or chemiluminescence-producing substrate reveals the position of each antibody- or antigen-producing cell as a point of color or light.

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