G ENERAL O VERVIEW FOR I MMUNOASSAYS

4.2.1 G ENERAL O VERVIEW FOR I MMUNOASSAYS

Immunoassays have been routinely used in medical and clinical settings for the quantitative determination of proteins, hormones, and drugs with a molecular mass of several thousand Daltons (Da). Immunoassay techniques including the enzyme- linked immunosorbent assay (ELISA) have also proven useful for environmental monitoring and human observational monitoring studies [6,19]. Common environ- mental pollutants (i.e., pesticides) are typically small molecules with a molecular mass of <1000 Da. This small size will not elicit antibody production. Small molecules (haptens) can be used for antibody production when conjugated to carrier molecules such as proteins. The small molecule of interest is usually modified to introduce a chemical moiety capable of covalent binding. The small molecule, or hapten, is then converted to an immunogenic substance through conjugation to the carrier molecule for antibody production. The design of a hapten greatly affects the selectivity and sensitivity of the resulting antibody. The distinguishing features of the small molecule must be preserved while introducing an additional chemical

group (i.e., –COOH, –OH, –SH, –NH 2 ) and linker chain or spacer arm for binding [5]. Hapten design, hapten synthesis, and antibody production are among the critical initial steps in developing immunoassays for small environmental pollutants.

A stepwise diagram for an ELISA is shown in Figure 4.1. This format is based on the immobilization of an antigen (i.e., the target analyte hapten conjugated to a

Wash

Ag is immobilized to the plate Ag/Ab mix is added to Ag-coated wells

Wash

Wash

Ab–Enzyme complex added Substrate added to produce color change

98 Analysis of Pesticides in Food and Environmental Samples protein) to a solid-phase support such as a test tube or a 96-well microtiter plate [20].

The sample extract for a microplate format (in a water-soluble solvent) and a solution of specific antibody (typically in phosphate-buffered saline [PBS] pH 7.4 containing 0.5% Tween 20) are added to the antigen-sensitized wells. The target analyte in solution and the immobilized antigen compete for binding sites on the specific antibody. The wells are rinsed with buffer to remove antibody not bound to the solid-phase antigen. The amount of antibody that can bind to the immobilized antigen on the plate is inversely related to the amount of analyte in the sample. A secondary antibody (species-specific that binds to the primary antibody) labeled with an enzyme (antibody-enzyme conjugate) is added to help visualize the presence of the bound primary antibody. Alkaline phosphatase and horseradish peroxidase are two commonly used enzyme labels. Another buffer rinse removes unbound excess enzyme-labeled secondary antibody. The addition of a chromogenic substrate pro- duces a colored end product that can be measured spectrophotometrically or kinet- ically for quantitation of analyte. This indirect competitive format is useful to support large observational studies due to its high sample throughput, adaptation to automa- tion, availability of commercial labels and substrates, and the high-performance level that can be achieved. For extremely high sample throughput capability, micro- titer plates containing 384 microwells can be used. In-depth details on how to develop antibodies and immunoassays, as well as data analysis are presented by Van Emon [2].

There are several permutations to the basic indirect competitive ELISA. Figure 4.2 depicts an immunoassay format using immobilized antibody and an enzyme-labeled tracer [21]. Analyte in the sample competes with a known amount of enzyme-labeled analyte for binding sites on the immobilized antibody. In the initial step, the antianalyte antibody is adsorbed to the side of a test tube or microtiter plate well. The analyte and an enzyme-labeled analyte are next added to the antibody-coated wells and competition for antibody binding occurs. After an incu- bation step, all unbound reagents are rinsed from the wells. Substrate is added for color development that is inversely related to the concentration of analyte present in the sample. This particular format is commonly used in immunoassay testing kits as

a few procedural steps are eliminated. However, this format does not have the convenience of commercially available reagents (i.e., enzyme-labeled secondary antibody) and requires the synthesis or labeling of either the analyte or hapten which may not be straightforward.