The CD cotinine kit is a competitive immunoassay designed and validated for the quantitative measurement of cotinine in saliva samples. This kit may be used to measure primary or secondhand exposure to nicotine. This kit is not for use in diagnostic procedures. It is intended only for research use in humans and some animals. A validated urine protocol is available on request.
Please read the complete kit insert before performing this assay. Failure to follow kit procedure and recommendations for saliva collection and sample handling may result in false values. For further information about this kit, its application, or the procedures in this insert, please contact the technical service team at CD or your local sales representative.
Contents of Kit
1. Microtitre Plate 1/96-well
2. Antiserum Contains: rabbit anti-cotinine antibody, buffer, preservative. 1 bottle/15 mL
3. Cotinine Standard In a saliva-like matrix. Contains: cotinine, buffer, preservative. 1 vial/500 µL
4. Cotinine Controls High, Low, in a saliva-like matrix. Ready to use. Contain: cotinine, buffer, preservative. 2 vials/250 µL each
5. Wash Buffer Concentrate (10X) Contains: phosphate buffer, detergent, preservative. 1 bottle/100 mL
6. Assay Diluent Contains: phosphate buffer, pH indicator, preservative. 1 bottle/60 mL
7. Cotinine Enzyme Conjugate Concentrate. Dilute before use with assay diluent. (See step 5 of procedure.) Contains: Cotinine conjugated to HRP, preservative. 1 vial/75 µL
8. TMB Substrate Solution Non-toxic, ready to use. 1 bottle/ 25 mL
9. 2 M Stop Solution Contains: sulfuric acid. 1 bottle/12.5 mL
All components of this kit are stable at 2-8°C until the kit's expiration date.
Intra-assay Precision (n=10): 4.5%-8.6%
Inter-assay Precision (n=8): 4.2%-9.0%
The lower limit of sensitivity was determined by interpolating the mean optical density minus 2 SDs of 10 sets of duplicates at the 0 ng/mL level. The minimal concentration of cotinine that can be distinguished from zero is 0.15 ng/mL.
The detection of exposure to tobacco smoke by measurement of cotinine is the preferred method. Nicotine is not considered a valid marker of smoking status due to its relatively short half-life (approximately 2 hours). By contrast, cotinine has an average half-life of 17 hours, and blood levels closely reflect the dose of nicotine absorbed from tobacco smoke. Saliva samples are easier to obtain, however, and saliva levels are highly correlated and used interchangeably with blood levels.
There is a clear need for inexpensive, accurate, quantitative, and noninvasive means of validating smoking status, measuring the immediate physiological consequences of individual differences and intraindividual change in smoking behaviors, and determining secondhand tobacco smoke exposure. CD has designed this research tool to provide biomedical researchers with a highly sensitive and reliable means to do so.
Cotinine levels in biologic fluids have been measured by chromatographic (GC or HPLC – sometimes coupled with mass spectrometry) and immunoassay methods. Chromatographic methods have the advantage of higher specificity and sensitivity, but EIA cotinine results have shown near perfect agreement with GC/MS confirmation of smoking status. Immunoassay methods also use smaller sample volumes than chromatography methods and they do not require extractions or other manipulations of the samples, making them easier to use in large-scale epidemiological studies and avoiding the need for specialized laboratories. Levels, however, may be higher with EIA since metabolites of cotinine, such as 3-OH-cotinine, are also measured.