A new general method to perform a noncompetitive immunoassay for low-molecular-mass analytes (less than 6000 Da) is described and checked using cortisol as a model system. The method is based on the use of a "polydentate ligand" (cortisol-poly(L-lysine) conjugate) able to block the antibody sites unoccupied by the analyte, followed by the replacement of an antibody-bound analyte by an enzyme-labeled analyte (cortisol-horseradish peroxidase), and permits the direct measurement of the analyte bound sites. The observed signal shows a near-linear correlation with the analyte concentration. The characteristics of interactions between the analyte and polydentate ligand with the specific antibody were studied to perform a preliminary evaluation of the noncompetitive immunoassay for cortisol. The noncompetitive assay was compared with a competitive immunoassay obtained under the same conditions and using the same reagents. The results of the experiments showed a lower detection limit for the noncompetitive model (0.15 ng mL-1 rather than 0.72 ng mL-1), emphasizing that the model is successful. Moreover, as the polydentate ligand is prepared from the same hapten used for the immunogen synthesis, this type of noncompetitive immunoassay appears generally applicable to all small molecules for which antibodies have been obtained.
A very selective polyclonal antiserum against ethyl 2-(4-phenoxyphenoxy)ethylcarbamate (fenoxycarb) was raised in a rabbit by immunization with a 2-(4-phenoxyphenoxy)ethanol hemisuccinate-bovine serum albumin conjugate. The antiserum was used to carry out a heterogeneous competitive enzyme immunoassay for the selective quantification of fenoxycarb at the ng ml 21 level. The effects on the assay performance of different pH and ionic strength values, as well as the presence of methanol as organic modifier in the diluent buffer formulation, were studied. The optimized calibration curve showed a sensitivity of 0.42 ng ml 21 . The assay selectivity was found to be good, with no significant cross-reactivity towards fenoxycarb-related pesticides such as chloroxuron, cypermethrin, diclofop, p,pA-DDT, difenoxuron, permethrin and pyriproxyfen.
The applicability of an ELISA for detection and quantification of benalaxyl in red wine samples is described. The study of the influence of this matrix on the reliability of the assay indicates that red wine samples require a rapid and simple cleanup step before ELISA assay. Recovery and precision of the method were evaluated by spiking red wine samples with benalaxyl in the 0.5-24 ng/mL range. Benalaxyl can be determined with good accuracy and precision up to 0. 5 ng/mL in starting red wine samples (detection limit of 0.13 ng/mL). No false negative or positive results were obtained. Authentic red wine samples were analyzed by ELISA and by RP-HPLC. The amounts of benalaxyl found by ELISA were in good agreement with RP-HPLC analysis.
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