A Simple and Rapid Enzyme-linked Immunosorbent Assay of Urinary Cotinine in Tobacco-smoke-exposed Subjects

Research Project

Project/Area Number	06670431
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Public health/Health science
Research Institution	Nara Medical University
Principal Investigator	YONEMASU Kunio Nara Medical University, Dept.Public Health, Professor, 医学部, 教授 (40028618)
Co-Investigator(Kenkyū-buntansha)	YOSHIOKA Nobuo Nara Medical University, Dept.Public Health, Research Associate, 医学部, 助手 (00254491) DOHI Yoshiko Nara Medical University, Dept.Public Health, Associate Professor, 医学部, 助教授 (50155628) KURUMATANI Norio Nara Medical University, Dept.Public Health, Assistant Professor, 医学部, 講師 (10124877)
Project Period (FY)	1994 – 1995
Project Status	Completed (Fiscal Year 1995)
Budget Amount *help	¥2,200,000 (Direct Cost: ¥2,200,000) Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000) Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywords	Tobacco smoke exposure / Cotinine quantitation / ELISA / Rapid and simple assay / Application to epidemiological investigation / 疫学調査 / 喫煙曝露量 / 尿中コチニン / 酵素免疫測定法
Research Abstract	Conventional methods of estimating tobecco smoke exposure have been based on questionnaire and thus the data connot be appropriate as such biological indicators. This research was, therefore, undertaken to develop a quantitative method of urinary cotinine (CN ; the major metabolite of cotinine) which is simple, rapid, accurate and furthermore good enough to apply to epidemiological investigation for masses of peopoe. Abrief outline of a developed method of quantifying CN with ELISA is as follows : known concentrations of CN-bovine thyroglobulin complex is fixed onto microtiter wells (lng/well), and aliquots of urine to be tested or of serial dilutions of standard CN-solutions (1-4000ng) together with an appropriate dilution of rabbit monospecific anti-CN-antisera are added. The reacted anti-CN-antibodies are quantified spectrophotometrically with peroxidase-labelled-anti-rabbit IgG-antibodies and ABTS.In the method, the lower limit of sensitivity is approximately 1ng and the measurable range is 1ng-4000ng. The method also allow to assay more than 40 urinary samples in duplicate with only one microtiter-plate. Urinary CN was quantified by using the developed method in 301 employees of a department store (165 non-smokers, 48 passive-smokers and 88 smokers). The median of the distribution of [25%, 75%tile] ng/ml of non-smokers, passive smokers and smokers are 10 [2.5,28] , 95 [40,145] , 3000 [1300,5000] ng/ml, respectively. The results showed that more than half of the self-reported non-smokers (the non-exposed) had been really exposed with tobacco smoke to the same levels of passive smokers. Thus the developed method has seemed to be more reliable and more appropriate to epidemiological investigation for masses of peopoe than conventional questionnaire-methods. The CN values also give us more objective and superior indication on the present state of the evaluation of the efficacy and guidance to create smoking-and non-smoking sections in workplace.