A monoclonal antibody-based enzyme-linked immunosorbent assay for human urinary cotinine to monitor tobacco smoke exposure

Abstract

Immunoassays for human urinary cotinine [(S)-(−)-cotinine], a major nicotine metabolite, are useful to monitor the degree of tobacco smoke exposure. However, practical monoclonal antibodies for measuring urinary cotinine are not widely available. Here we generated a monoclonal antibody against a newly prepared cotinine–albumin conjugate and developed an enzyme-linked immunosorbent assay (ELISA). Splenocytes from a BALB/c mouse that had been immunized with the conjugate were fused with P3/NS1/1-Ag4-1 myeloma cells, and a hybridoma clone secreting the anti-cotinine antibody, Ab-CT#45 (IgG1κ; amino acid sequences of the variable domains were determined), was established. This antibody was immobilized on microplates to generate an ELISA system in which biotin-labeled cotinine was used as the tracer molecule. After competitive reactions with analyte, the bound biotin residues were monitored colorimetrically with peroxidase-labeled streptavidin. This ELISA produced dose–response curves for cotinine ranging from 0.40–100 ng per assay and was able to discriminate nicotine, cotinine N-oxide, cotinine N-glucuronide (cross-reactivity was each <0.5%, taking cotinine as 100%), (3′R, 5′S)-3′-hydroxycotinine O-glucuronide (0.6%) and (R, S)-norcotinine (1.5%). Another major nicotine metabolite, (3′R, 5′S)-3′-hydroxycotinine, showed 8.4% cross-reactivity, but an analytical recovery test indicated that this metabolite did not result in a significant overestimation of the urinary cotinine levels. The present ELISA has been validated to allow the urine specimens to be “directly” (without pretreatment) measured in order to screen and monitor tobacco smoke exposure, and if necessary, to enable cotinine alone to be more specifically examined after the urine specimens are subjected to a simple chloroform extraction.