Competitive 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. 

A series of monoclonal antibodies were generated that can bind dimetridazole (269) and other nitroimidazole drugs used in veterinary medicine. An extraction procedure was developed for these nitroimidazoles that is compatible with a competition ELISA method, based on binding of these antibodies to the drugs. As little as 1 ng of 269 could be detected in turkey muscle by this method552. 

In 1993, another immunoassay for the detection of monensin was developed but, unfortunately, was never applied to biological material (91). Quite recently a competitive ELISA and a compatible extraction procedure suitable for screening monensin in poultry liver samples was described (92). In this assay, a polyclonal antiserum raised against a monensin-transferrin conjugate and prepared via an acid anhydride intermediate (93) was used. Significant cross-reactivity with other polyethers commonly used by the broiler industry, such as maduramicin, lasalocid, salinomycin, and narasin, was not found. A detection limit of 3 ppb could be readily attained when liver samples were submitted to extraction with aqueous acetonitrile, partitioning between aqueous sodium hydroxide solution and a hexane-diethyl either mixture, evaporation of the organic phase, and reconstitution in ethanol/sodium acetate solution. 

Other anticoccidials for which immunoassays have been developed include the polyethers salinomycin and lasalocid, and the quinazoline drug halofuginone. Concern over the potential toxicity of salinomycin has led to development of two competitive ELISAs for detecting and measuring salinomycin residues in poultry tissues. In the first ELISA (96), 16 monoclonal antibodies were prepared and evaluated for their ability to produce a rapid and low-cost screening procedure for residues of salinomycin in poultry liver. In the second (97), the antibodies produced showed cross-reactivity with narasin but not with lasalocid, madura-... 

ELISA. The focus of this chapter, the competitive ELISA, usually is the preferred choice when a DAS ELISA is not available because it provides greater specificity than the direct ELISA, making it more reliable for the diagnostic procedures it is being used for (7,2). 

In competitive ELISA, the procedure used is very similar to that described above for RIA the antigen coupled to the enzyme is bound to an antibody immobilised on... 

This chapter deals with relatively simple ways to use control charts to monitor the performance of ELIS As. A rinderpest competition ELISA, for the estimation of antibodies in serum samples, is used to demonstrate the methods. This assay is available in kit form. Constant evaluation of the use of the kit is part of what is called internal quality control (IQC). Figure 1 shows an overview of the ELISA scheme described in this chapter. The details of the procedure, which involves plotting the data graphically (charting methods), are explained herein. The objectives of charting data are as follows ... 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

About 10% of serum proteins are immunoglobulins (Igs). After immunization, the specific antibodies produced are about 1"C5% of this fraction, so the required Ig (in ELISA) may be from 0.1 to 2.5% of the total protein in a serum. Some assays are favored by the relatively crude fractionation of serum to obtain Igs, e.g., for use in binding to plates in trapping (sandwich assays) to avoid competition for plastic binding sites by other serum proteins. Several methods for separation of Igs are available for use in ELISA. These procedures are suitable for polyclonal antibodies but not necessarily for mAbs. The isolation of total Igs as compared to the purification of specific Igs, is relatively simple. 

The procedures for production of specific antibodies and their application in a competitive inhibition ELISA (Enzyme-Linked Immunosorbent Assay) are discussed in detail in the preceding chapter (Vanderlaan et al., this volume). In addition, other comprehensive overviews of the immunoassay development process in the pesticide field are available in general, a... 

The evaluation of a number of immunoassay diagnostic kits was undertaken to determine their usefulness in a regulatory analytical laboratory environment in the food, feed and pesticide areas. Four rapid enzyme immunoassay tests for the detection of aflatoxin residues at the 20 ppb level in animal feeds were compared to the official HPLC procedure. In the pesticide area, a commercial pentachlorophenol competitive inhibition assay for residues in water was investigated as to its applicability to poultry and pork liver matrices. In addition, an ELISA screening procedure for the herbicide fusilade was developed. Modifications were incorporated into the rapid immunoband 1-2 Test procedure for the detection of motile Salmonella in various food and animal feed products resulting in quicker analysis than the standard culture method. Also, a comparative evaluation of a Quik-Card Test for sulphamethazine drug residues in pork urine, liver and muscle tissue, is described. 

Figure 6. Time course for 3-Cys-A adduct formation in liver and serum proteins. B6C3F1 male mice which had been fasted overnight were administered acetaminophen (400 mg/kg). The procedures for the preparation of the protein samples and the competitive A-B ELISA for quantitation of the acetaminophen are described in (1 ). The data points are mean + SEM. All data on acetaminophen adduct formation in serum from 2 to 24 hours are significantly different from control values. The acetaminophen adduct formation in liver protein from one hour to eight hovu s is significantly different (P 0.05) from control values however, the acetaminophen adduct formation at the 12 and 24 hoiu time points are not significantly different from the untreated controls (Reproduced from Ref. 19).

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