Enzyme-linked immunosorbent assay with antibody-coated plates

Even more popular than dot blots are microtiter plate assays, so-called ELlSAs (enzyme-linked immunosorbent assay), in which antibody or antigen is loaded into the depression of polyvinyl chloride or polystyrene plates (Kemeny 1994). The depressions are then further coated with antibody, antigen, and enzyme-conjugated antibody in a defined sequence. The antigen is detected via an enzymatic color reaction (Table 6.2). Many companies (Nunc, Flow, Costar, Falcon) offer a palette of products such as 8- or 12-channel pipettes, automatic washing devices, ELISA readers, and so on that make life easier for the friends of ELISA. 

Competition Enzvme-Linked Immunosorbent Assay. A competition enzyme-linked immunosorbent assay (cELISA) was developed to quantify the amount of heptachlor in solution and to evaluate the ability of the antibodies to distinguish among various cyclodiene insecticides and related chemicals. Microtiter plates were coated with 0.25 ng/well hept-BSA (100 p.l/well of a 2.5 ng/mL solution of hept-BSA in distilled water was allowed to evaporate onto the bottoms of the wells at 37° C). The plates were then blocked with DB as described above. Competitors (analytical standards dissolved in methanol) were added to the DB such that the resulting solution was 50% methanol. An aliquot (200 pL) of this competitor-dilution buffer solution was added to an antigen-coated well. Then, the amount of competitor was serially diluted down the microtiter plate, so each well contained 100 iL of competitor in a 50% methanol-dilution buffer solution. Next an equal volume of dilution DB 100 ng of anti-heptachlor monoclonal antibody was added to each well. Thus, each well contained 200 )xl of a 25% methanol solution in DB, antibody and competitor. Plates were incubated for 1 h at 37° C and then processed as described above. 

Bioassay-based or enzyme-linked immunosorbent assay (ELISA) methods have been applied in epitope mapping [18, 19]. In this approach, different antigens are coated separately on the surface in different weUs of the analytical plate followed by incubation with a specific antibody that is linked to an enzyme. When the enzyme s substrate is added to the solution, a subsequent reaction can produce a detectable signal. This approach, however, is mostly limited to the characterization of linear epitopes. ELISA can also be applied to multisite binding analysis, where one antibody is attached to the surface, the antigen is bound, and the ability of the second antibody to bind to the attached antibody-antigen... 

Enzyme-linked immunosorbent assays (ELISAs) in kit form are most widely used giving relatively rapid and inexpensive methods for multispecies identification. A typical format for such an ELISA is to coat different strips of the normal 12 x 8-well plate with antisera formed against serum albumin of the various species of interest. An extract of the meat product is added to the antibody-coated wells, incubated to ensure antibody binding of the serum albumin, and, after washing, a second antibody coupled with enzyme is introduced. The sandwich is visualized by addition of a substrate to the enzyme. ELISAs have been developed, also, for meat species identification in cooked meat products. These ELISAs are quite specific and sensitive ( 1% of each species can be detected) but are qualitative, or at best, semiquanti-tative. 

It is possible to perform an enzyme-linked immunosorbent assay (ELISA) using a streptavidin-coated 96-well plate the primary antibody, biotinylated-rabbit-anti-streptavidin, specifically recognizes the avidin linked to the helicate as the bioprobe. The detection limit is better than that with commercially available organic dyes such as antistreptavidin-FICT (FiB3). 

An enzyme-linked immunosorbent assay (ELISA) was performed as described previously. Briefly, each well of a 96-well microtiter plate was coated with 100 pi of the indicated concentration of sample in PBS, and incubated for 2 hr. The wells were washed three times with PBS containing 0.05% Tween 20 (washing buffer), then blocked with 0.5% gelatin in PBS for 1 hr. After washing 3 times, the wells were incubated for 1 hr with 100 pi of the primary antibody including monoclonal anti-CML antibody (2G11 1 pg/ml), monoclonal anti-CEL antibody (CEL-SP 5 pg/ml) and monoclonal anti-GA-pyridine... 

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