A general method to perform a noncompetitive immunoassay for small molecules

A new general method to perform a noncompetitive immunoassay for low-molecu lar-mass analytes (less than 6000 Da) is described and checked using cortis ol as a model system. The method is based on the use of a "polydentate liga nd" (cortisol-poly(L-lysine) conjugate) able to block the antibody sites un occupied by the analyte, followed by the replacement of an antibody-bound a nalyte by an enzyme-labeled analyte (cortisol-horseradish peroxidase), and permits the direct measurement of the analyte bound sites. The observed sig nal shows a near-linear correlation with the analyte concentration. The cha racteristics of interactions between the analyte and polydentate ligand wit h 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 p olydentate ligand is prepared from the same hapten used for the immunogen s ynthesis, this type of noncompetitive immunoassay appears generally applica ble to all small molecules for which antibodies have been obtained.