ELISA, procedure

Enzyme-linked immunosorbent assay (ELISA). Li and Li developed an ELISA procedure for imidacloprid to determine its residues in coffee cherry and bean extracts. A 25-g amount of sample extracted with 300 mL of methanol and 1% sulfuric acid (3 1, v/v) for 3 min. An aliquot of the sample extract (0.5 mL) is mixed with 1 mL of water and a gentle stream of nitrogen is used to evaporate methanol. The solution is then extracted with 1 mL of ethyl acetate, the extract is reconstituted in 1 mL of PBST (phosphate-buffered saline containing 0.05% Tween 20) and competitive ELISA is performed to quantify imidacloprid in the extract. Eor methanol extracts of coffee cherries and beans fortified with imidacloprid at 0.5 mgL recoveries of imidacloprid by the ELISA method were 108 and 94, respectively. 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

Although numerous research articles have been written describing the preparation and use of antibody conjugates with P-gal, the enzyme remains a minor player in ELISA procedures. Less than 1 percent of all commercial ELISA products utilize this enzyme. 

A sensitive ELISA procedure was developed for the determination of TNT (221) and other nitroaromatic compounds. TNT can be detected within the range of 0.02-20 ng/L in water samples462. A simplified immunofiltration prepacked portable device for field screening tests of TNT in water and soil was also developed. A quantitative color response to concentrations of TNT in the range 1-30 ng/L in water and 50-1000 pg/g in soil was demonstrated463. 

An earlier attempt by Berger and Berger (29) to develop an enzyme-linked immunosorbent assay (ELISA) procedure with sheep-ant i-ciguatoxin was unsuccessful. In part, this may be attributable to the approaches taken and to the insufficient number of samples of documented toxic and non-toxic fishes examined. Except that the putative toxic fish samples came from Tahiti, evidence for their toxicity was not presented in the report (29). 

A. Larsson, An ELISA procedure for the determination of protein G-binding antibodies, J. Immunol. Methods, 135 (1990) 273-275. 

If quantitative analysis of antibody-phage binding is desirable, it is possible to perform an ELISA procedure using purified phage as antigen. 

In addition to the further increase in sensitivity, the advantages of microplate-based detection methods are manifold The direct digital readout of a conventional microplate-reader allowed for easy processing of multiple samples the compatibility to standard ELISA procedures ensures a robust and simple handling, and therefore also a significant decrease in method... 

Several qualitative and quantitative immunochemical methods and their application to the analysis of environmental samples have been described for OP insecticides, a family that includes widely used pesticides such as azinphos-ethyl/methyl, dichlorvos, fenitrothion or fenthion, malathion, mevinphos, and parathion. Mercader and Montoya202 produced monoclonal antibodies against azinphos-methyl and developed an ELISA that was used for the analysis of water samples from different sources, reaching detectability levels near 0.05 pg I. Watanabe et al.203 reported the production of polyclonal antibodies and ELISA procedures to analyze fenitrothion in river, tap, and mineral water (LOD = 0.3 pg L ). Banks et al.204 produced polyclonal antibodies against dichlorvos, an organophosphate insecticide used for stored grain, which also cross-reacts with fenitrothion. Nishi et al.205 reported the first immunoassay for malathion. Residues of this insecticide have... 

Immunosensors have made a great contribution in the field of androgenic steroid detection, giving detection limits comparable to those obtained with standard ELISA procedures. Several electrochemical immunosensors have been developed for detecting testosterone, methyltestosterone,... 

Bi- or multi-functional reagents have been used to link enzymes to other proteins. These include various carbodiimides, bisdiazotized amines, cyanuric chloride and glutaraldehyde. Enzymes linked to rabbit IgG antibodies from sheep, goats, and horses are commercially available and greatly facilitate the ELISA procedure. The preparation of the antigen and the development of the corresponding rabbit antibody (IgG fraction) have been described previously. 

The ELISA procedure recently has been used for the analysis of parathion (31). Since this procedure has considerable potential a more detailed description of the analysis of parathion is in order. The conjugation procedure using amino parathion (AP) was described earlier (Fig. 3, Rn 9), and this conjugate was then administered to rabbits for development of a population of specific antibodies (Abi) against BSA or AP. Abi demonstrated immunological activity only for the hapten when AP was conjugated to rabbit serum albumin (RSA). This antigen (RSA-AP) was rendered insoluble via attachment to the polystyrene surface of microtiter plates under basic conditions (Fig. 6.1). 

The procedure gave good reproducibility as expressed in the coefficient of variation (CV%) of results of between-run (6.2-8.6) and within-run (4.8-6.5) variations. Accuracy of the ELISA procedure was tested by comparing results of parathion analysis in extracts of fortified and field samples with results obtained by a GLC method. Correlation coefficients ranged in almost all cases between 0.93-0.99. 

The ELISA procedure for the analysis of parathion as described above requires nearly eight hours, although many samples can be simultaneously assayed. However, incubation times can be shortened to one-half hour, in most cases, resulting in only a 10% reduction in sensitivity. Also the polystyrene microtiter plates containing bound RSA-AP can be mass produced and stored in a freezer. Since the enzyme-linked antibody can be purchased, the limiting factor of the applicability of the ELISA procedure, as well as the RIA procedures, for other pesticides is the development of the antiserum to the pesticide. 

Cost Effectiveness. As with the other advantages of immunochemical analysis, cost may be quite variable. Reagent costs for several automated systems have been estimated at under 1.25 per sample. The cost is obviously much lower for less sophisticated assay systems, especially if some reagents are prepared in house. A major consideration is the expense of new instrumentation. For dedicated or automated instrumentation for either RIA or ELISA procedures, the cost may be 50-100,000. However, most analytical laboratories already have the basic instrumentation needed for immunoassays. Moderate sensitivity can be obtained through the use of numerous procedures such as radial immunodiffusion and hemagglutination. These procedures require no expensive equipment or reagents and they may be very useful in areas where equipment acquisition or maintenance is a problem. 

Indirect ELISA Procedure Using Antigen-Coated Plates... 

Coat microtiter plates with immunoglobulins (1-20 /rg/ml) purified from antiserum as described37 and diluted in carbonate buffer (see ELISA procedure above).28... 

Perform three washing steps with PBS-T. Subsequent incubation steps are conducted as in the ELISA procedure above. The antiimmunoglobulin conjugate must be specific for the second antibody (e.g., rabbit antibody) used to react with the trapped antigen and should not react with the first antibody (e.g., chicken antibody) used for coating the plates. 

Several variations of indirect ELISA procedures have been described.25-38... 

Incubate with antigen as in ELISA procedures above. 

Standard radio-immunoassay procedures are applied, nowadays mainly solid phase assays which can be rapidly performed and evaluated, they are also several enzyme-linked immunoassay (ELISA) procedures from commercial suppliers. It is recommended to perform an internet check for the most appropriate method at the time of the study (for example DSL 2005). Methods have been described for triiodothyronine (Nejad et al. 1975, Chopra etal. 1972a, Larsen 1972a, 1972,) and for thyroxin (Chopra etal. 1971, Chopra 1972). The use of assays based on thyroxin binding globulin (Chopra et al. 1972b) is no longer recommended and cannot be applied to the rat, because the rat does not have this binding protein. However, for the human, measurement... 

Mercaptopyridine derivatized agarose beads were used as thiophilic adsorbents for antibodies in radioimmunoassay (RIA) and ELISA procedures. 

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