Nicotine Antibodies: Comparison of Ligand Specificities of Antibodies Produced against Two Nicotine Conjugates

Abstract

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...

Enhanced immunogenicity of a bivalent nicotine vaccine

“…Presumably the two nicotine immunogens stimulated distinct populations of B cells even though both elicited the production of nicotine-specific antibodies. A related finding was reported by Castro et al [28], who reported that antibodies produced using a 6-aminonicotine immunogen did not crossreact with a 2-aminonicotine hapten. However, this study used 125 I-tyrosine methyl ester linked to nicotine through urea, rather than nicotine, as the radioligand for cross reactivity determination, which complicates interpretation of cross-reactivity data, and did not test the cross reactivity of the antibodies produced by the 2-aminonicotine immunogen.…”

Nicotine Vaccines to Assist with Smoking Cessation

“…[67] Due to its small molecular weight, nicotine itself is not immunogenic; in order to trigger an immune response, nicotine or a structurally similar hapten needs to be linked to a carrier protein,[68,69] thus producing a conjugate vaccine. The success of an immunological strategy relies on several characteristics, including but not limited to (i) immunogenicity of the vaccine; (ii) affinity of antibodies; and (iii) specificity of antibodies.…”

Biomarkers Derived from Nicotine and its Metabolites: A Review

“…Cotinine has also been determined using enzyme immunoassay (EIA) (147,148). Immunoassays have been used to determine nicotine concentrations in amniotic fluid (149), blood plasma and serum (150)(151)(152)(153)(154)(155), cervix mucus (153,156), hair (157)(158)(159), meconium (160), saliva (154), seminal plasma (155), and urine (154,161). Cotinine concentrations have been determined by immunoassays in amniotic fluid (149,162), blood plasma and serum (17,145,(153)(154)(155)(163)(164)(165)(166), cervix mucus (153,156); follicular fluid (167)(168)(169), hair (157)(158)(159)(160)(161)(162)(163)(164)(165)(166)(167)(168)(169)…”