Immunoassays for proteins alkylated by nicotine-derived N-nitrosamines

Abstract

Polyclonal antibodies recognizing the pyridyloxobutyl (POB) moiety of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were produced in rabbits immunized either with POB-bovine albumin or POB-Sepharose. The POB intermediates necessary to modify the protein were generated by alkaline (pH 9.0) treatment of the synthetic precursor 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone. In a competitive enzyme linked immunoabsorbent assay (ELISA), 70 pmole NNK inhibited 50% of the binding of the anti-POB antibodies to POB-protein absorbed on microtiterplates. This 50% inhibition varied from 70 pmole to 200 nmole using a series of NNK analogues, depending on the integrity of the POB moiety. Immunological techniques initiated in this study detect NNK-protein conjugates or measure the quantity of POB groups liberated upon alkaline or acid treatment of NNK modified protein.

Synthesis of 4-[2-Aminoethyl(nitrosamino)]-1-pyridin-3-yl-butan-1-one, a New NNK Hapten for the Induction of N-Nitrosamine-Specific Antibodies

“…Because of the 300-fold mass difference, direct binding of NNK to mAbs was not measurable by surface plasmon resonance; therefore, the relative specificity of some structurally and metabolically related molecules were ranked by competition ELISA. In contrast to the antibodies induced against the pyridyl-oxobutyl moiety by Talbot et al (34), our antibodies recognized NNK and, even better, NNAL. The competition capacity of the pyridyl ring (IC 50 ) 400 µM The side chain in position 3 of the pyridine ring improved the recognition as exemplified by the efficient binding of ethyl nicotinate (IC 50 ) 49-80 µM); however, both mAbs showed some differential sensitivity to functional groups on the side chain.…”

“…Because of the 300-fold mass difference, direct binding of NNK to mAbs was not measurable by surface plasmon resonance; therefore, the relative specificity of some structurally and metabolically related molecules were ranked by competition ELISA. In contrast to the antibodies induced against the pyridyl–oxobutyl moiety by Talbot et al , our antibodies recognized NNK and, even better, NNAL. The competition capacity of the pyridyl ring (IC 50 = 400 µM or 320 µM for 3-picoline) corresponded to 25–40% of the competition capacity of NNK (IC 50 = 80 µM or 160 µM) for P9D5 or P7H3 calculated from the OD using the dynamic range of the assay.…”

“…Also, as a result of the low reactivity of the endpoint metabolites (HPB, PB, PBCA, and the keto acid), these would not compete effectively for the antibodies against activated NNK metabolites. Finally, in contrast to the antibodies induced by the pyridyl–oxobutyl hapten of Talbot et al , our antibodies would not only bind to NNK but also to NNAL, the two most carcinogenic species of the metabolic cascade.…”

“…Thus, our synthetic approach and the final conjugate vaccine differ considerably from that of Talbot et al (34), who used the 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone, lacking in particular the tertiary amine with its nitroso group and bovine serum albumin as a protein carrier.…”

Immunochemical Detection of Sulfur Mustard Adducts with Keratins in the Stratum Corneum of Human Skin

“…Information on immunochemical assays for the detection of protein adducts is only scarcely available in the literature. In general, the antibodies have been generated by using adducted keyhole limpet hemocyanin or albumin as an antigen ( − ). Such an approach was followed in one of our previous studies by using sulfur mustard treated hemoglobin as antigen.…”