Competitive ELISA using

Other attempts have been made to detect BPA at a low concentration range. Thus Kodaira et al. [274] analyzed BPA in urine samples with an assay that showed a working range between 0.5 and 5 pg L The assay was validated by HPLC. DeMeulenaer et al. [275] developed an indirect competitive ELISA using PAbs obtained from chicken egg yolk, but the assay achieved an IC50 value of only 570 pg L-1. 

Lindbladh C, Persson M, Buiow L, et al. The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion. Biochem Biophys Res Gommun 1987 149 607-14. 

Maucher, J.M. and Ramsdell, J.S., Ultrasensitive detection of domoic acid in mouse blood by competitive ELISA using blood collection cards, Toxicon, 45, 607, 2005. 

Manager B, Solve M, Eriksen H, Brogen CH (1994). Bovine 3-lactoglobulin in hypoallergenic and ordinary infant formulas measured by an indirect competitive ELISA using monoclonal and polyclonal antibodies. Food Agric. Immunol, 6(l) 73-83. 

Table I. Mouse Competitive ELISA Using Anti-retronamine...

Shelby RA, Kelley VC (1992) Detection of ergot alkaloids from Claviceps species in agricultural products by competitive ELISA using a monoclonal-antibody. J Agric Food Chem 40 1090-1092... 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

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. 

Regarding commercially available immunochemical kits, we could mention the Charm ROSA Enrofloxacin Test that detects ciprofloxacin and enrofloxacin equally (see Table 4) and the 5101ERFXlp test. This last one is a direct competitive ELISA, which uses MAbs and has a LOD of 3 ng g1 in tissues. Some other companies do have antibodies available as reagents for different applications such as Biodesign International and QED Bioscience Inc. 

Regarding sensors, Draisci et al. [100] reported the development of an electrochemical competitive ELISA for the detection of erythromycin and tylosin in bovine muscle. They used MAbs against these two macrolides and the activity... 

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. 

Vemuri et al.17 looked at the effects of various cryoprotectants, freezing rates, and buffer systems on the shelf-life of lyophilized recombinant alphar antitrypsin (rAAT). Alpharantitrypsin (AAT) is labile in solution therefore, a more stable presentation was required. A competitive ELISA was used to measure total AAT in a sample. The AAT in the sample competed with HRP-labeled AAT for binding to the specific antibody. A stable formulation containing lactose as a cryoprotectant was found that maintained the protein s specific activity. 

RIAs are highly sensitive and quantitative, capable of detecting small amounts of Ag or Ab. As a result, they are often used to measure the quantities of hormones or drugs present in a patient s serum. In this case, RIAs are performed in a manner similar to the competitive ELISA. The presence of the hormone in the serum sample inhibits binding of the radiolabeled hormone. Thus, the amount of radioactivity present in the test is inversely proportional to the amount of hormone in the serum sample. A standard curve using increasing amounts of known concentrations of the hormone is used to determine the quantity in the sample. 

An indirect competitive ELISA has been also developed for the determination of streptomycin and dihydrosticptomyciri in milk (24). Prior to the analysis, the milk sample was skimmed and treated with oxalic acid. The antiserum was raised in rabbits using streptomycin linked to a bacterial protein as the antigen. To perform the test, microtiter plates were coated with streptomycin, and antiserum and milk samples were mixed to be added in the wells where they were incubated for 1 h. Depending on the amount of residues in the sample, more or less antibody remained available for binding to the streptomycin coat. A pig antirabbit antibody-enzyme conjugate was subsequently added and incubated for 90 min. Using a suitable substrate, streptomycin and dihydrostreptomycin could be detected down to 1.6 ppb, whereas quantification could be made possible up to 100 ppb when samples were used undiluted. 

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. 

Halofuginone can be also analyzed in chicken serum by a competitive ELISA developed on the basis of monoclonal antibodies (99). In this study, a serum matrix effect that afforded a higher sensitivity for the detection of halofuginone in chicken serum than in assay buffer or in highly diluted serum was observed. The sensitivity of the ELISA improved when used in more concentrated serum. 

Figure 17.5—The steps in a competitive ELISA assay. Several assays of this type are used in clinical analysis.

Fig, 2, Competitive ELISA.corves obtained using primary ailatoxins(left) and some aHatoxin metabolites (right). 

This method is further divided into two major types. One type is competitive ELISA, which can be used for the analysis of both hapten and macromolecule the other is noncompetitive sandwich-type ELISA, which is only used for divalent and multivalent antigens. Two major types, i.e., direct competitive ELISA (dC-ELISA) and indirect competitive ELISA (inC-ELISA), are used most commonly in food analysis. 

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). 

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