Submitted to: International Conference on Agri-Food Antibodies
Publication Type: Abstract Only
Publication Acceptance Date: 10/5/2001
Publication Date: N/A
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 monoclonal antibodies (Mabs) to 4,4'-dinitrocarbanilide (DNC), the active component of fthe coccidiostat nicarbazin. The synthesis for the hapten (p-nitro-cis- 1,2-cyclohexanedicarboxanilic acid (PNA-C)) that was used in a conjugate with bovine serum albumin as the plate coating antigen is described. Mabs were isolated that compete with nicarbazin, and they had an isotype of IgMk. Due to the lack of water solubility of nicarbazin or DNC, some organic solvents were required in the assay buffer to achieve solubility. Both DMF (3%) and ACN (10%) were added to the assay buffer for ELISA studies with nicarbazin and related compounds. The Nic 6 monoclonal antibody IC35 value (IC percentage was the center of the inhibition curve) for nicarbazin was estimated to be 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). It 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.