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Nicotine and cotinine are the compounds most frequently quantified by three different types of quantitative immunoassays based on radioactivity, spectrophotometry, and fluorescence measurements. Applications of immunoassay to other nicotine metabolites are described in this chapter. Cotinine is a compound commonly assayed to determine nicotine exposure from various sources. As a biomarker, cotinine possesses several desirable properties. It has a considerably longer half life than nicotine and is usually found in higher concentrations in the physiological fluids of the average smoker [6,48]. It is a relatively stable metabolite. Cotinine is absent (or present in only minute amounts) in tobacco and in the nicotine replacement devices designed to deliver nicotine per se , i.e., chewing gum, skin patches, nasal sprays, and inhalers. Its presence in physiological fluids clearly indicates that nicotine absorption and metabolism occurred. Immunoassays are continually modified to make them more sensitive, rapid, and easier to perform. For example, the RIAs for nicotine and cotinine are much more sensitive today with the availability of labeled nicotine of higher specific activity; (-)-[N-methyl-3H]nicotine currently in use has a specific activity of 60–87 Ci/mmol compared with (-)-[3H]nicotine, specific activity 2.4 Ci/mmol, used earlier. ELISAs are more sensitive when substrates with increased color yields are used. These devices (immunosensors) have the potential for increased sensitivity and rapid, simultaneous analysis of large numbers of test samples.

L. Levine , H. Fujiki , H. B. Gjika and H. Van Vunakis . A radioimmunoassay for palytoxin. Toxicon26, 1115–1121, 1988.—Palytoxin, labelled with 125I-Bolton-Hunter reagent on its terminal amino group, bound specifically to rabbit anti-palytoxin. The extent of binding increased progressively with repeated immunizations. After absorption of the rabbit IgGs with a goat anti-rabbit IgG, binding was reduced greater than 95%. For 50% inhibition of binding in the 125I-palytoxin-anti-palytoxin reaction 0.27 pmoles of unlabelled palytoxin was required. Maitotoxin, teleocidin, okadaic acid, debromoaplysiatoxin and 12-O-tetradecanoylphorbol-13-acetate, when tested at 10–100-fold higher concentrations than palytoxin did not affect binding. Palytoxin's serologic activity was stable after 60 min exposure to 100°C and after 60 min exposure to 0.1 N HCl at 50°C, but its capacity to stimulate the arachidonic acid metabolism of rat liver cells was reduced after the 60 min exposure to 0.1 N HCl treatments at 35°C or 0.01 N HCl at 50°C. The average binding constant (K0) as determined by separation of antibody-bound palytoxin from free palytoxin by the double antibody technique was 4.9 × 109 M−1 at 0°C. This apparent average association constant increased with increasing temperature suggesting that palytoxin's epitope, most likely hydrophilic, is bound to H2O and the H2O is displaced before binding to the antibody's paratope.

L. Levine , H. Fujiki , H. B. Gjika and H. Van Vunakis . Production of antibodies to palytoxin: neutralization of several biological properties of palytoxin. Toxicon 25, 1273 – 1282, 1987. — Palytoxin stimulated arachidonic acid metabolism (in bovine aorta endothelial and smooth muscle cells, rat keratinocytes, porcine aorta endothelial cells and rat liver cells), hemolyzed rat erythrocytes and was lethal to mice when administered intraperitoneally. Serum from rabbits immunized with a conjugate in which palytoxin was covalently bound to bovine albumin through its free amino group neutralized these biologic activities of palytoxin. Ninety-nine per cent of the neutralizing activity of the immunized rabbit serum was removed after precipitation of the rabbit IgG with a goat anti-rabbit IgG.

Serum and salivary cotinine levels were measured in 327 smoking and nonsmoking participants in a study of the health effects of marijuana with and without tobacco. These individuals had no reason to misrepresent their current tobacco-smoking status. The sensitivity, specificity, and predictive values positive and negative of the cotinine levels in distinguishing self-reported current tobacco smokers from nonsmokers was high (88-100%) and essentially the same for both fluids. Agreement between self-report and cotinine levels was not influenced by the presence or absence of marijuana smoking. A good correlation was found between serum and salivary cotinine levels in self-reported tobacco smokers (r = 0.84, p less than 0.001). Mean average levels were 279 +/- 144 ( +/- standard deviation) ng/ml for serum and 360 +/- 195 ng/ml for saliva. In a separate group of seven tobacco smokers, cotinine levels in saliva were found to be essentially independent of salivary flow rate. An analogous relationship has been observed by others for various compounds that are filtered to saliva from the blood. This may explain the close relationship observed between serum and salivary cotinine levels, and the observation made by others that the half-life of salivary cotinine is similar to that of serum cotinine.

Antibodies specific for N6(methylnitroso)adenosine have been produced in rabbits and a sensitive radioimmunoassay was developed. The nitroso group is immunodominant; 50% inhibition of the binding of [3H]N6(methylnitroso)adenosine to antibody was obtained with 9.6 pmoles of N6(methylnitroso)adenosine and 200 nmoles of N6-methyladenosine. Adenosine was essentially inactive. After nitrosation, N6(methylnitroso)adenosine can be detected only in those RNA molecules known to contain N6-methyladenosine.

L. Levine , H. Fujiki , K. Yamada , M. Ojika , H. B. Gjika and H. Van Vunakis . Production of antibodies and development of a radioimmunoassay for okadaic acid. Toxicon 26, 1123–1128, 1988.—An okadaic acid immunogen, prepared by conjugation of okadaic acid to bovine albumin with carbodiimide, was used to immunize two rabbits. The rabbits responded by producing antibodies that neutralized okadaic acid's stimulation of arachidonic acid metabolism and this neutralization increased during the course of immunization. The immune sera bound 3 H-okadaic acid and this binding also increased with repeated immunization. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 99%. The binding of okadaic acid to the antibodies in one antiserum was inhibited by as little as 0.2 pmoles of unlabelled okadaic acid. The apparent association constant for binding with this antiserum was 4.17 × 10 9 M −1 (35°C). Maitotoxin, teleocidin, 12- O -tetradecanoylphorbol-13-acetate, aplysiatoxin, palytoxin and breve-toxin B when tested at 29, 228, 168, 169, 3.7 and 112 pmole levels, respectively, did not inhibit binding. The serologic and biological activities of okadaic acid after incubation for 60 min in 0.01 N HCl at 35°C or at 100°C at pH 7.2 were unaffected.

For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N- succiny -[ 3 H] phhosphoamino acids. When the unlabeled N-succinl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivatively respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 × 1010 M−1 for phosphotyrosine, 0.825 and 6 × 108 M−1 for phosphothreonine, and 0.150 and 2 × 108 M−1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytolasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.

The purpose of this study was to assess nicotine regulation among "heavy" and "light" smokers. Previous studies supporting the nicotine regulation model of smoking behavior have suggested that smokers compensate for a reduction in the amount of nicotine available in their cigarette by altering smoking frequency, puff volume, or other aspects of smoking topography. However, little is known about a smoker's decision to smoke a specific cigarette, and the concurrent changes in their blood nicotine. Manipulation of nicotine levels in the blood could play a critical role in smoking maintenance, by regulating the extent and quality of the CNS effects of smoking. In this study, 24 heavy and light smokers (cotinine above or below 260 ng/ml) smoked high- (1.0 mg) or low- (0.5 mg) dose nicotine cigarettes while watching non-stressful movies. Blood nicotine was assessed before and after smoking a preload and free operant cigarette. The results showed that blood nicotine levels after smoking the free operant cigarette were significantly more consistent (lower standard error) for the heavy smokers, following a low dose, as opposed to a high-dose preload. Light smokers showed a non-significant trend towards being more consistent when the high-dose nicotine preload was used. This suggests that heavy smokers may have maximized their dose of nicotine whenever available nicotine was in relatively short supply (low dose condition). However, light smokers may have minimized their exposure when available nicotine was relatively more plentiful (high dose condition).

Antibodies to D-lysergic acid have been produced in rabbits and guinea pigs and a radioimmunoassay for the hapten was developed. The specificity of this lysergamide-antilysergamide reaction was determined by competitive binding with unlabeled lysergic acid diethylamide (LSD), psychotomimetic drugs, neurotransmitters, and other compounds with diverse structures. LSD and several related ergot alkaloids were potent competitors, three to seven times more potent than lysergic acid itself. The N , N -dimethyl derivatives of several compounds, including tryptamine, 5-hydroxytryptamine, 4-hydroxytryptamine, 5-methoxytryptamine, tyramine, and mescaline, were only about ten times less effective than lysergic acid, even though these compounds lack some of the ring systems of lysergic acid. The pattern of inhibition by related compounds with various substituents suggests that the antibody receptor site recognizes structural features resembling the LSD molecule. In particular, the aromatic nucleus and the dimethylated ethylamine side chain in phenylethylamine and tryptamine derivatives may assume in solution a conformation resembling ring A and the methylated nitrogen in ring C of LSD. Among the tryptamine derivatives, a large percentage of the most potent competitors are also psychotomimetic compounds.

A teleocidin A-2 derivative, 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2, induced ornithine decarboxylase in mouse skin and inhibited the specific binding of [3H]12-O-tetradecanoyl-phorbol-13-acetate to a mouse skin particulate fraction with a potency that was weaker than that of teleocidin A-2 and stronger than that of (-)-indolactam-V. To obtain antibodies, 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2 was covalently linked to bovine albumin with a carbodiimide and the resulting conjugate used for immunization of rabbits. Antibodies directed toward teleocidin were produced as measured by neutralization of teleocidin's capacity to stimulate arachidonic acid metabolism in rat liver cells (the C-9 cell line). An 125I-labeled ligand was prepared by reaction of the same derivative with radiolabeled Bolton-Hunter reagent. The antibodies bound this radiolabeled hapten, and the binding increased progressively with repeated immunizations. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 95%. The binding of the 125I-labeled ligand to the antibodies of one rabbit had an apparent average association constant of 2.84 x 10(9) M-1 at 0 degrees C. The serologic specificity of the antiserum was characterized by measuring the inhibition of binding by several teleocidins of varying structure as well as by other tumor promoters and toxins. The rank order of inhibitory activity expressed as concentration required for 50% inhibition (IC50) was for 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2'(0.56 pmol) greater than teleocidin A-1 (6.5 pmol) greater than or equal to teleocidin A-2 (7.3 pmol) greater than (-)-indolactam-V (3.7 nmol) greater than teleocidin B-4 (13 nmol). Maitotoxin, aplysiatoxin, palytoxin, mezerein, okadaic acid and 12-O-tetradecanoylphorbol-13-acetate did not inhibit at the levels tested.

Radioimmunoassays capable of detecting pseudouridine, N 2 -dimethylguanosine, and 7-methylguanosine at picomole levels were developed. The antibodies to the nucleoside-human serum albumin conjugates recognize the modified ribose linked to the ϵ-amino group of lysine. The relative serological activities of the different nucleosides in the pseudouridine anti-pseudouridine-human serum albumin reaction depend upon the presence of the ribose ϵ-aminocaproate moiety in the radiolabeled antigen and/or the competing unlabeled nucleoside.

Publisher Summary Prostaglandins (PG's) are widely distributed in mammalian tissues and biologic fluids. As PG's are low molecular weight compounds, they must be linked covalently to a carrier protein or polypeptide to render them immunogenic. Conjugation of the PG to poly(L-lysine) is achieved by the use of a water-soluble carbodiimide. The dehydration reaction results in the formation of an amide bond between the carboxyl groups of the PG's and e-amino groups of poly(L-lysine). As poly(L-lysine) is a positively charged macromolecule, it must be complexed to a negatively charged macromolecule (that is, succinylated hemocyanin) for successful production of antibodies. The poly(L-lysine) conjugate is complexed with the succinylated hemocyanin by adding the latter dropwise to a solution of the conjugate (approximately 200–500 μg of succinylated hemocyanin are added per 1 mg of conjugate). Prior to immunization, the complex is emulsified with an equal volume of complete Freund's adjuvant (Difco Laboratories, Detroit, and Mich.). One milligram of the prepared antigen is sufficient for injection of two rabbits or monkeys.

Antibodies directed toward 12-L-hydroxyeicosatetraenoic acid (12-L-HETE) were generated in rabbits by immunization with conjugates of 12-L-HETE and human serum albumin. The concentration of antibodies was determined by incubating immune plasma with 12-L-HETE that had been covalently linked to a solid support, washing the 12-L-HETE support, and measuring the quantity of bound antibodies by reaction with [125I] Protein A. The addition of 0.5 ng-10 ng of fluid-phase 12-L-HETE to the standard mixture of solid-phase 12-L-HETE and anti-12-L-HETE plasma inhibited by 21–80% the binding of antibodies and consequently of [125I]Protein A to the solid support. The 12-OH function positioned between two double bonds was the immunodominant determinant of this antigen-antibody reaction, but the carboxyl function also was recognized. This radioimmunoassay was used to detect and quantitate 12-L-HETE resolved by high pressure liquid chromatography.

Following a period of overnight deprivation, 58 smokers participated in a 90-min laboratory assessment in which they viewed a non-stressful movie and smoked two 0.5-mg nicotine-containing cigarettes. The first cigarette was given to all subjects following 25 min of adaptation and baseline. The next cigarette was provided at their request, which occurred 9–12 min later. “Heavy” and “light” smokers were grouped according to their average morning cotinine values, which fell above or below 250 ng/ml, respectively. The results showed that, relative to their baseline, heavy and light smokers experienced about the same level of post-smoking change in blood nicotine, heart rate and blood pressure. However, heavy smokers showed a significantly greater delta from baseline in post-smoking measures of epinephrine, norepinephrine, tension reduction and increase in vigor enhancement. A strong and consistent correlation was observed between post-smoking increases in epinephrine, tension reduction and increased vigor.