Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 28, 2001
Publication Date: August 21, 2001
Interpretive Summary: Nicarbazin is a drug that is placed in feed to control coccidiosis, a disease that occurs during production of broiler chickens. We have developed antibodies that recognize nicarbazin. Antibodies are substances that are produced by the immune system in response to foreign substances which enter the body. Once the antibody to a foreign substance is isolated, it can be used in a method to detect the presence of that foreign substance. The antibodies that we isolated to detect nicarbazin may be used in an easy-to-use field test to make direct checks for appropriate levels of nicarbazin in animal feed. This would ensure that the poultry received the necessary dose to prevent coccidiosis, but not more. This capability would help USDA Food Safety and Inspection Service (FSIS) be sure that producers have complied with FDA regulations for preventing residues in meats.
Technical Abstract: An enzyme-linked immunosorbent assay (ELISA) was developed for the coccidiostat nicarbazin. Based on insights previously obtained from computer assisted molecular modeling, p-nitrosuccinanilic acid (PNA-S) was selected as a viable hapten candidate, and was used to produce antibodies to 4,4'-dinitrocarbanilide (DNC), the active component of the coccidiostat nicarbazin. Synthesis of the hapten p-nitro-cis-1,2- cyclohexanedicarboxanilic acid (PNA-C) with bovine serum albumin used for the 96-well plate coating conjugate is described. Monoclonal antibodies having an isotype of IgM kappa compete with nicarbazin. Due to poor water solubility of nicarbazin and DNC, some organic solvents were required in the assay buffer to achieve solubility. Both Dimethylformamide (3%) and acetonitrile (10%) were added to the assay buffer for ELISA studies with nicarbazin and related compounds. The monoclonal antibody designated Nic 6 had an IC35 value (IC percentage was the center of the inhibition curve) for nicarbazin of 0.92 nmol/mL, with a limit of detection at 0.33 nmol/mL nicarbazin. Nic 6 exhibited high cross-reactivity for PNA-S (immunizing hapten) and PNA-C (plate coating hapten). Nic 6 also had high cross-reactivity for 3-nitrophenol, 4-nitrophenol, and 1-(4-chlorophenyl)- 3-(4-nitrophenyl)urea. However, Nic 6 had little or no cross-reactivity with fifteen other related compounds. The non-active component in nicarbazin, 2-hydroxy-4,6-dimethylpyrimidine, exhibited no cross- reactivity with Nic 6, nor with the other isolated monoclonal antibodies.