Enzyme-linked-immunosorbent microtiter plate

EIAs are more desirable for the measurement of agrochemicals than enzyme-linked immunosorbent assays (ELISAs) for several reasons. EIAs are easier to run, require minimal liquid transfers, and are completed in brief time frames, approximately 40 min for tube assays to 2.5 h for microtiter plate assays. In contrast, ELISAs are more complex, have many steps involving transfer of reagents, and require 6-8 h to complete. Most commercially available immunoassays utilize the EIA format. 

Fig. 8. Schematic presentation of a enzyme linked immunosorbent assay (ELISA). An antigen ( ) is immobilized on the surface of a microtiter plate and incubated with its antibody (abl). A second antibody (ab2) with a covalently linked enzyme ( , e.g., horseradish peroxidase) binds to the primary one and catalyzes a color reaction with its enzyme. All incubations are separated by washing steps...

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

Enzyme-linked immunosorbent assay reader or UV/VIS spectrophotometer for microtiter plates (e.g., Dynatech, Chantilly, VA, USA). 

The enzyme-labeled antibody can be evaluated by enzyme-linked immunosorbent assay. Immobilize the appropriate antigen on the wells of a microtiter plate or strip (at a concentration of 2-10 ]Xg/ mL), incubate various dilutions of the conjugate for a few hours, wash the wells, add substrate, and measure the amount of product formed (see Chapter 15). This approach may also be used for monitoring conjugate purification in chromatography fractions. 

Pomes, M.L., E.M. Thurman, and D.A. Goolsby (1996). An evaluation of a microtiter-plate enzyme-linked immunosorbent assay method for the analysis of triazine and chloroacetanilide herbicides in storm run-off samples. In J.M. Van Emon, C.L. Gerlach and J.C. Johnson, eds., Environmental Immunochemical Methods Perspectives and Applications. ACS Symposium Series 646. Washington, DC American Chemical Society, pp. 170-182. 

Enzymes are currently the most widely used and investigated labels for immunoassays, because a single enzyme label can provide multiple copies of detectable species. This catalytic amplification results in immunoassay detection limits that rival those of radioimmunoassay without the storage and disposal problems associated with radioisotopes. Enzyme immunoassays label either ligands or antibodies with enzyme, and enzyme activity in bound or free fractions is measured. Heterogeneous immunoassays employing enzymatic labels have been named enzyme-linked immunosorbent assays (ELISAs). ELISA methods usually employ antibody immobilized onto the wells of polystyrene microtiter plates, and may be... 

Assay optimization. An optimization step not always taken, but nonetheless important to the success of any immunochemical method of analysis is the selection of materials, such as test tube or plastic plates, which maximize assay performance. An example of this is the selection of 96-well microtiter plates for enzyme-linked immunosorbent assay (ELISA) which give maximum protein binding capacity and minimum interwell variability (28>32 also Harrison, R.O. Nelson, J.O. J. Immunoassay. submitted). The type of microtiter plate may be the most important single determinant of ELISA performance and this selection should not be made carelessly. Significant error may also occur in the reading of assays performed in 96-well microtiter plates, due to alignment errors of automatic plate readers undetected in normal use (Harrison, R.O. Nelson, J.O. J. Immunoassay. submitted Harrison, R.O. Hammock, B.D. J. Assoc. Off. Anal. Chem.. submitted), and a plate reader test should allow further reduction of error. These steps should be taken before a major investment in time, effort, or money is made in an assay system which may later be found to be less than acceptable. 

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. 

Figure 5 Schematic representation of the most widely used noncompetitive biologieal immunoassay, the enzyme-linked immunosorbent assay (ELISA), (a) Antibody, selective for analyte, is immobilized on microtiter plate well surface (b) sample added (c) analyte in sample binds to antibody while other compounds in matrix remain in solution (d) sample solution containing nonbound molecules discarded and wells washed (e) analyte remains bound to antibody (f) second antibody nzyme conjugate added (g) conjugate binds to bound analyte (h) solution containing nonbound conjugate discarded and wells washed (i) conjugate remains bound 0 enzyme substrate added (k) substrate S converted to colored or fluorescent product P at a rate which is proportional to the amount of bound enzyme and hence to the concentration of analyte in the sample.

Enzyme-linked immunosorbent assay (ELISA) methods are widely used in medical and food analysis laboratories and it seems appropriate that such methods could be used for the detection of mycotoxins in foods. In the direct ELISA method the antibody is bound to a solid surface, such as a microtiter plate, and it is necessary to prepare a conjugate of the mycotoxin to be analyzed with the enzyme, such as phosphatase or peroxidase, to be used for color development. The sample or standard is mixed with the enzyme-linked mycotoxin and the two allowed to compete for the antibody bound to the surface. After washing away soluble material, the... 

Although unlike RIA, enzyme immunoassays can be carried out in homogeneous systems without a separation step (based on the change in enzyme activity during the immune reaction), in practice, heterogeneous (enzyme-linked immunosorbent assay) methods are much more frequently used. The antibody, or in the case of the double-antibody method the second antibody, is immobilized, either covalently or by coating enzyme multiplied immunoassay technique (EMIT). This can be done on the walls of microtiter plates. After the immunogenic reaction, the enzyme activity, which is the equivalent of radioactivity in RIA systems, can be measured by suitable photometric methods on the microtiter plates themselves. 

Enzyme-linked immunosorbent assay (ELISA) ELI-SAs detect and amplify antigen-antibody reactions by using covalently bound enzyme-antibody molecules. The presence of the enzyme (indicating presence of the antigen) is detected by the addition of the appropriate substrate. Detection systems are usually designed to produce a color change that can be quantified by a microtiter plate reader. 

The important trend of assay parallelization (similarly to the ELISAs (enzyme linked immunosorbent assays) performed in microtiter plates) has increased the use of miniaturized electrodes and, film electrodes, especially those fabricated by screen-printed technology are widespreading. By their importance, these will be discussed in a separate section. 

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... 

Immunological methods make use of antibodies as analytical tool for detecting a plethora of clinical, environmental, and food-relevant analytes. The special features that had made immunoassay widely increased in the last decades are the highly sensitivity and specificity of the antibody-antigen interaction. Although enzyme-linked immunosorbent assay (ELISA), currently performed in microtiter plates is the most common technique, a variety of assay types can be performed depending of the analytes or samples. 

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