Improved 125I radioimmunoassay for cotinine by selective removal of bridge antibodies.
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.
D000818:Animals; D001952:Bridged-Ring Compounds / Q000276:immunology; D055598:Chemical Phenomena; D002621:Chemistry; D003367:Cotinine / Q000031:analogs & derivatives / Q000276:immunology / Q000652:urine*; D003429:Cross Reactions; D006241:Haptens; D006433:Hemocyanins; D006801:Humans; D007106:Immune Sera; D007163:Immunosorbent Techniques; D007457:Iodine Radioisotopes; D009538:Nicotine; D011760:Pyrrolidinones / Q000652:urine*; D011817:Rabbits; D011863:Radioimmunoassay; D012907:Smoking; D014316:Tritium
D001952:Bridged-Ring Compounds; D006241:Haptens; D007106:Immune Sera; D007457:Iodine Radioisotopes; D011760:Pyrrolidinones; D014316:Tritium; C043966:N-(4-hydroxyphenethyl)cotinine carboxamide; D009538:Nicotine; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D003367:Cotinine