The enzyme-linked immunosorbent assay (ELISA) method for nicotine metabolites determination in biological fluids

The aim of this study was to present the usefulness of enzyme-linked immunosorbent assay (ELISA) in nicotine metabolites determination in urine and serum samples from active smokers and compare it with the reference to thin layer chromatography (TLC) with densitometry. The specific anti-cotinine antibodies were obtained from rabbit sera after sequential immunization with 4'-carboxycotinine-hemocyanine conjugate. The immunoaffinity chromatography technique with the use of self-prepared cotinine-aninohexyl-sepharose bed enabled the isolation of the specific anti-nicotine metabolites antibodies from the antiserum. Affinity of isolated antibodies to cotinine was passively immobilised on ELISA plates and competition between nicotine metabolites in samples and tracer (horseradish peroxidase-cotinine conjugate) was applied. After the washing stage the enzymatic activity of solid-phase-bound peroxidase was determined. For calibration cotinine perchlorate solutions in appropriate matrix were used. Determination ranges for serum and urine samples were from 3 to 1500 and from 3 to 5000 ng/mL, respectively. Precision within-run and between-run was below 8.7 and 11.3%; mean recovery of cotinine was 100.59% from serum and 88.56% from urine samples. The ELISA method, used in determination of the main nicotine metabolites showed high accuracy and sensitivity. However, this method was less specific than the reference technique (TLC). The high correlation coefficients, r>0.9, between the results of determined nicotine metabolites in urine by means of ELISA and TLC with densitometry confirmed the possibility of the application of ELISA method to practical monitoring of tobacco smoke exposure in large population groups.