Development of an enzyme linked immunosorbent assay for the determination of phenothiazine drugs in meat and animal feeds.

In this study, 2-chlorophenothiazine was used to synthesize a hapten for production of monoclonal antibody. The obtained monoclonal antibody showed high crossreactivities to chlorpromazine, promethazine and perphenazine, and showed low crossreactivities to acepromazine and fluphenazine. After evaluation of three coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the five phenothiazines in animal feeds and the residues of chlorpromazine, promethazine and perphenazine in meat. The crossreactivities to the five analytes were in a range of 2.4%-98%. The limits of detection for the five drugs in feeds were in a range of 0.1-3.0 µg g-1, and that for chlorpromazine, promethazine and perphenazine in meat were in a range of 0.5-0.8 ng g-1. Their recoveries from standards fortified blank samples (chicken, pork and feeds) were in a range of 74.1%-96.5% with coefficients of variation of 6.4%-15.1%. Therefore, this method could be used as a rapid screen tool to determine phenothiazine drugs in animal feeds and animal derived foods.

Synthesis of hapten and production of a monoclonal antibody against a derivative of L-hydroxyproline, a special amino acid in hydrolyzed animal protein.

China's government has prohibited the addition of simply hydrolyzed animal protein from solid leather waste into milk. The objective of this study was to produce a monoclonal antibody against L-hydroxyproline, a special amino acid in hydrolyzed animal protein. L-hydroxyproline was derivatized with N-acetylsulfanilyl chloride and 5-chlorovaleric acid to synthesize the haptens HP1 and HP2. Then, two immunogens from the two haptens were prepared to produce the antibodies. Results showed that only HP1 was able to stimulate the animal immune system and generate the specific antibody to L-hydroxyproline (as the formation of HP1). The obtained monoclonal antibody from HP1 and the heterologous coating hapten HP2 were incorporated into a competitive indirect enzyme linked immunosorbent assay (ELISA) to determine the antibody's specificity and sensitivity. The IC(50) and the limit of detection for HP1 were 0.16 µg/mL and 0.05 µg/mL respectively. The antibody showed low crossreactivity to parental L-hydroxyproline and showed negligible crossreactivity to D-hydroxyproline and other amino acids. The monoclonal antibody was therefore suitable for the development of an immunoassay to monitor the simply hydrolyzed animal protein from solid leather waste in foodstuffs with L-hydroxyproline as the target analyte.