The level of cotinine in biological specimens, such as serum, urine, and saliva, measured by gas or liquid chromatography is the most validated and reliable indicator of exposure to tobacco smoke. However, chromatographic methods are not always suitable for all types of situations.

We validated a commercially available enzyme-linked immunosorbent assay ( ELISA ) that uses a polyclonal antibody to cotinine as a practical alternative to chromatographic methods.

The cotinine antibody cross-reacts to 3-hydroxycotinine (3HC) and its glucuronide, thus generating a value for immunoreactive (IR) cotinine, which is a complex comprising cotinine, 3HC, and 3HC-glucuronide. The levels of IR cotinine in the urine of kindergarten children closely correlated with those of cotinine measured by gas chromatography–mass spectrometry (GC–MS) and reflected the smoking behavior of their parents more precisely than cotinine levels determined by GC–MS.

Our findings showed that the cotinine-based ELISA can be a practical biomarker of exposure to tobacco smoke.