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Clinical Chemistry 31: 118-121, 1985;
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Clinical Chemistry, Vol 31, 118-121, Copyright © 1985 by American Association for Clinical Chemistry

Improved 125I radioimmunoassay for cotinine by selective removal of bridge antibodies

GJ Knight, P Wylie, MS Holman and JE Haddow

We describe an 125I-based RIA for cotinine, the major metabolite of nicotine. The slope of the dose-response curve was quite shallow (6-8% change in binding per doubling dose), resulting in between-assay CVs of 15 to 20%. This effect occurred because the radioligand formed by linking a cotinine derivative to tyramine manifested greater affinity for the anti-cotinine antibodies than did cotinine itself. We absorbed the serum with a derivative of nicotine coupled to the carrier protein via a chemical bridge similar to that used to form the cotinine/carrier protein immunogen. An RIA in which we used such absorbed serum showed a significantly increased slope of the dose-response curve (11-13% change in binding per doubling dose), and between-assay CVS were only 6 to 8%. We suggest that this improvement results because absorption removes anti-bridge antibodies directed against the chemical-bond common to the cotinine/carrier-protein immunogen and to the cotinine/tyramine radioligand.


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