Conjugates between bovine serum albumin and (R,S)-2-aminonicotine were produced, and these conjugates were employed in rabbits and goats for the production of nicotine antibodies. In the assay of nicotine, an 1251-tyrosine methyl ester derivative of (R, S)-6-aminonicotine was employed as radioligand. The antibody-bound derivative was separated from the free derivative by charcoal adsorption (0.5 % charcoal, 0.1 % dextran T-70, 0.1 % bovine serum albumin, pH 7.3). Among the twenty five nicotine derivatives and metabolites examined, (R, S)-6-aminonicotine gave the highest cross-reaction. Cross-reaction with cotinine, a major mammalian metabolite of nicotine, was less than 0.1 % for both the rabbit-derived and goat-derived antisera. Cross-reaction by other metabolites, such as (5')-nicotine-"-oxide, (5')-nornicotine, and N-methylpyrrolidine was less than 1 %.The antibodies produced were thus highly specific to nicotine. The radioimmunoassay for nicotine showed a maximum sensitivity of 10 ng/ml in 5O-pl plasma samples for both antisera. After the smoking of a single cigarette (1.2 mg nicotine content in mainstream) the peak blood plasma level of nicotine in the subjects varied from 20 -104 ng/ml, and high levels of nicotine were not necessarily found in heavy smokers.Interest in the metabolism of nicotine has been stimulated during the past two decades by an increased consideration of the possible role of nicotine as a reinforcer [I] in the habitual use of tobacco and by many studies on the short half-life of nicotine in human blood plasma [2-41. In addition, some of the metabolites of nicotine appear [5-71 in various instances physiologically to oppose or enhance the effects of nicotine. These and other factors have combined to focus our attention on effective methods for determining the kinetic factors in the biodisposition of nicotine. Basic to a general understanding of this biodisposition has been a general need for rapid and accurate determinations of nicotine and its metabolites in biological material. This need has been met in part by gas chromatography and combined gas chromatography/mass spectrometry. Limitations and advantages of these and other methods have been previously reviewed [8,9]. Attention in our laboratories [lo, 111 and those of others [12-151 has been directed, therefore, to various immunological techniques for the rapid determination of nicotine.The low molecular weight of nicotine and the extremely limited tendency of the alkaloid to form stable molecular linkages with many macromolecules has dictated strongly against the possibility of producing nicotine antisera of useful titer by employing the parent alkaloid as the immunogen, and functionalization of nicotine has been necessary. In functionalizing nicotine prior to coupling with macromolecules for antibody production, both the pyridine ring and the pyrrolidine ring of the alkaloid are available. In particular, our early and recent attention has been devoted to 2-aminonicotine and 6-aminonicotine as functionalized haptens, since, as oth...

Enzyme immunoassay for cortisol was developed using beta-galactosidase as an enzyme label and m-maleimidobenzoyl derivatives of cortisol, i.e. cortisol-21-m-maleimidobenzoate (CT-MB) and cortisol-21-hemisuccinate conjugated with m-maleimidobenzoic acid through p-phenylenediamine linkage (CHS-MB), as the haptens coupled to sulfhydryl groups of the enzyme. This enzyme-coupling procedure was highly efficient; over 90% of the enzyme was labeled while full enzyme activity was retained. CHS-MB-beta-galactosidase conjugate showed high immunoreactivity to antibody produced against cortisol-21-hemisuccinate-bovine serum albumin but showed poor displacement with the added cortisol. CT-MB-beta-galactosidase conjugate, however, showed not only a high immunoreactivity to the antibody but also displaced well with cortisol, showing maximum sensitivity of 1 microgram/dl with a 20-microliter sample size. Modification around the linkage of cortisol derivative resulted in high sensitivity to cortisol. Cross-reactions to cortisol-21-acetate, cortisone, and corticosterone were 150%, 10%, and 7%, respectively. The accuracy, precision and correlation with RIA of this method were satisfactory for clinical application.

We synthesized the m-maleimidobenzoyl derivative of digoxigenin-3-0-succinate (through a p-phenylenediamine bridge) as a hapten derivative directed towards coupling to sulfhydryl grouos of beta-galactosidase. Prepared enzyme conjugate had about 97% of the enzyme labeled with the hapten derivative while retaining full enzyme activity. The enzyme immunoassay for digoxin we prepared showed a maximum sensitivity of 30 pg per assay (c.v. = 3%) with minimal cross-reaction with digotoxin (3.8%). Our method for hapten conjugation to beta-galactosidase is highly efficient and is simple and easily replicated.