Group-selective antibodies based fluorescence immunoassay for monitoring opiate drugs

A novel carboxylic acid derivative of monoacetylmorphine (MAM-COOH) was synthesized and conjugated with bovine serum albumin (BSA) for generating polyclonal antibodies against the target molecule heroin and its major metabolites. The conjugate was characterized by fluorescence spectroscopy, polyacrylamide gel electrophoresis, and mass spectrometry to confirm the extent of haptenization of the carrier protein. A high titer ( 1: 64,0000) of antibody was obtained by using the conjugate with an optimum protein/hapten molar ratio of 1:100. The generated antibody showed good binding affinity with heroin and its metabolites monoacetylmorphine (MAM) and morphine. The relative affinity constant (Kaff) of the antibody was 3.1 x 10(7) 1 mol(-1), and the IC50 values obtained for heroin, MAM, morphine, and codeine were 0.01, 0.013, 0.012, and 0.014 ng ml(-1), respectively. A fluorescence-based competitive inhibition immunoassay procedure was developed for the estimation of heroin and its major metabolites in standard and biofludic samples over a concentration range up to 0.01 ng ml(-1) with good signal reproducibility (p < 0.05). The method can be used as a convenient quantitative tool for the sensitive screening of major metabolites of heroin in biological samples.