A sensitive enzyme immunoassay for measuring cotinine in passive smokers.
BACKGROUND
Both active smoking and passive exposure to tobacco smoke are major risk factors for cardiovascular, pulmonary, and oncological diseases. The serum level of cotinine, a major proximate metabolite of nicotine, reflects active or passive exposure to tobacco smoke. However, currently available enzyme-linked immunosorbent assays (ELISAs) for cotinine have limited sensitivity, and a high-throughput quantification of the severity of passive exposure to tobacco smoke has not been possible thus far.


METHODS
We generated a phage display of combinatorial antibody library, from which we selected a recombinant antibody against cotinine, developed a sensitive ELISA using this antibody, and evaluated the method in a clinical setting and an animal model.


RESULTS
The limits of detection and the lower limit of quantification were 31pg/mL and 1ng/mL cotinine, respectively. The intra- and inter-assay precisions based on three quality control samples were 3.8-13.5% and 14.0-15.0%, respectively. No significant interference from nicotine, trans-3'-hydroxy cotinine, tobacco alkaloids, or other serum components was found. When we applied our ELISA to serum samples from 36 volunteers, the serum cotinine levels were clustered into two groups, which exactly corresponded to their smoking behavior and this ELISA yielded reproducible and accurate results, which were comparable to those of LC/MS in a split assay. In animal studies, we were able to distinguish between rats injected with a nicotine dose equivalent to that of passive exposure to tobacco and rats without exposure.


CONCLUSIONS
The competitive ELISA described here is useful for the detection and quantification of the severity of risk of passive smoking.
Park
Sunyoung
S
Department of Biochemistry and Molecular Biology, Seoul National University School of Medicine, Republic of Korea.
Lee
Do-Hoon
DH
Park
Jae-Gahb
JG
Lee
Yong Tae
YT
Chung
Junho
J
D000818:Animals; D001483:Base Sequence; D002853:Chromatography, Liquid; D003367:Cotinine / Q000097:blood*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D013058:Mass Spectrometry; D009538:Nicotine / Q000008:administration & dosage; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D014028:Tobacco Smoke Pollution
D017931:DNA Primers; D014028:Tobacco Smoke Pollution; D009538:Nicotine; D003367:Cotinine