One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity.

Immunoassays for cotinine, a major nicotine metabolite, in the urine are useful for monitoring the degree of tobacco smoke exposure. However, hybridoma-based anti-cotinine antibodies lack sufficient binding affinity to perform practically sensitive measurements, and thus most cotinine assays still rely on polyclonal antibodies. Here, we describe the generation of a mutant single-chain Fv fragment (scFv) that was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary cotinine levels in passive smokers. A "wild-type" scFv (scFv-wt) with a Ka value of 2.7 × 107 M-1 (at 4 °C) was prepared by linking the VH and VL domains in a mouse anti-cotinine antibody. "One-shot" random mutagenesis on the scFv-wt gene by error-prone PCR generated mutant scFv genes, which were expressed on phage particles. Repeated panning directed toward mutants with slower off-rates selected scFv clones that showed improved sensitivity in an ELISA system. One of these mutants (scFv#m1-54) with five amino acid substitutions showed more than a 40-fold enhanced Ka (1.2 × 109 M-1 at 4 °C) and, thus, was used to monitor human urinary cotinine. A limited amount of soluble scFv was reacted with urine specimens (or cotinine standards) at 4 °C for 120 min in microwells on which cotinine residues had been immobilized. The midpoint of the dose-response curves under optimized conditions (0.27 ng/assay) was more than 100-fold lower than the ELISA results obtained using scFv-wt. The limit of detection (8.4 pg/assay) corresponded to 0.17 ng/mL urinary cotinine, which was satisfactorily low for testing the threshold levels for passive smoke exposure. The assay values for volunteers correlated with the values determined using a commercial assay kit. This study evidently showed the potential of a molecular breeding approach, in which simple in vitro evolution might generate superior antibody reagents as cloned proteins, overcoming the limited molecular diversity inherent to conventional immunization-based antibodies.

A simple physiologically based pharmacokinetic model evaluating the effect of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans.

Physiologically based pharmacokinetic (PBPK) modeling was applied to investigate the effects of anti-nicotine antibodies on nicotine disposition in the brains of rats and humans. Successful construction of both rat and human models was achieved by fitting model outputs to published nicotine concentration time course data in the blood and in the brain. Key parameters presumed to have the most effect on the ability of these antibodies to prevent nicotine from entering the brain were selected for investigation using the human model. These parameters, which included antibody affinity for nicotine, antibody cross-reactivity with cotinine, and antibody concentration, were broken down into different, clinically-derived in silico treatment levels and fed into the human PBPK model. Model predictions suggested that all three parameters, in addition to smoking status, have a sizable impact on anti-nicotine antibodies' ability to prevent nicotine from entering the brain and that the antibodies elicited by current human vaccines do not have sufficient binding characteristics to reduce brain nicotine concentrations. If the antibody binding characteristics achieved in animal studies can similarly be achieved in human studies, however, nicotine vaccine efficacy in terms of brain nicotine concentration reduction is predicted to meet threshold values for alleviating nicotine dependence.

An aptamer-antibody complex (oligobody) as a novel delivery platform for targeted cancer therapies.

Aptamers have recently emerged as reliable and promising targeting agents in the field of biology. However, their therapeutic potential has yet to be completely assessed due to their poor pharmacokinetics for systemic administration. Here, we describe a novel aptamer-antibody complex, designated an "oligobody" (oligomer+antibody) that may overcome the therapeutic limitations of aptamers. To provide proof-of-principle study, we investigated the druggability of oligobody in vivo using cotinine conjugated t44-OMe aptamer, which is specific for the sequence of pegaptanib, and an anti-cotinine antibody. The antibody part of oligobody resulted in extended in vivo pharmacokinetics of the aptamer without influencing its binding affinity. Moreover, the aptamer of oligobody penetrated deeply into the tumor tissues whereas the anti-VEGF antibody did not. Finally, the systemic administration of this oligobody reduced the tumor burden in a xenograft mouse model. Together, these results suggested that our oligobody strategy may represent a novel platform for rapid, low-cost and high-throughput cancer therapy.

In vitro and in vivo application of anti-cotinine antibody and cotinine-conjugated compounds.

The combination of a high-affinity antibody to a hapten, and hapten-conjugated compounds, can provide an alternative to the direct chemical cross-linking of the antibody and compounds. An optimal hapten for in vitro use is one that is absent in biological systems. For in vivo applications, additional characteristics such as pharmacological safety and physiological inertness would be beneficial. Additionally, methods for cross-linking the hapten to various chemical compounds should be available. Cotinine, a major metabolite of nicotine, is considered advantageous in these aspects. A high-affinity anti-cotinine recombinant antibody has recently become available, and can be converted into various formats, including a bispecific antibody. The bispecific anti-cotinine antibody was successfully applied to immunoblot, enzyme immunoassay, immunoaffinity purification, and pre-targeted in vivo radioimmunoimaging. The anti-cotinine IgG molecule could be complexed with aptamers to form a novel affinity unit, and extended the in vivo half-life of aptamers, opening up the possibility of applying the same strategy to therapeutic peptides and chemical compounds.

Bispecific Her2 × cotinine antibody in combination with cotinine-(histidine)2-iodine for the pre-targeting of Her2-positive breast cancer xenografts.

PURPOSE: Cotinine has optimal characteristics as a hapten for pre-targeted radioimmunotherapy (PRIT). This study was performed to evaluate the applicability of cotinine/anti-cotinine antibody to PRIT. METHODS: We developed and prepared a tandem, single-chain, variable fragment Fc fusion protein [tandem single-chain variable fragment (scFv) Fc fusion protein] that is reactive to both human epidermal growth factor receptor 2 (Her2) and cotinine. Its simultaneous reactivity to Her2 and cotinine was tested in an enzyme-linked immunosorbent assay (ELISA) and two radioimmunoassays (RIA) employing Her2-coated RIA tubes and a Her2-overexpressing cell line. For in vivo imaging, mice bearing Her2-positive tumors were injected with a mixture of tandem scFv Fc fusion and (125)I-cotinine-conjugated histidine dipeptide ((125)I-cotinine peptide). After a delay, (125)I-cotinine peptide was injected again. RESULTS: ELISA and RIA results showed that tandem scFv Fc fusion protein successfully bound to both Her2 and cotinine. In single-photon emission computed tomography (SPECT), the complex of tandem scFv Fc fusion protein and (125)I-cotinine peptide was localized to Her2-positive tumor xenografts in mice 4 h after the first injection. Enhanced radioactivity at the site of the Her2-positive tumor lesion was monitored 1 h after the second injection. CONCLUSIONS: With these findings, we conclude that the tandem scFv Fc fusion protein and cotinine hapten system have the potential to be applied in PRIT.

Different binding modes of free and carrier-protein-coupled nicotine in a human monoclonal antibody.

Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 Å resolution. Nicotine is almost completely (>99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge-charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation-π interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine-carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine-carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine-linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180°. This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained.

Secondhand smoke exposure and inflammatory markers in nonsmokers in the trucking industry.

BACKGROUND: Few studies have directly assessed the association of secondhand smoke (SHS) with cardiovascular disease-related inflammatory markers, and the findings are inconsistent. OBJECTIVES: We assessed the association between SHS exposure and the inflammatory markers high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and soluble intercellular adhesion molecule-1 (sICAM-1) in 199 nonsmoking U.S. trucking industry workers. METHODS: Participants provided blood samples either by mail (blood drawn at local health care provider near home) or at the work site (blood drawn by research staff on-site) and completed a health and work history questionnaire at the time of blood draw. Exposure to SHS was measured by plasma cotinine concentrations. We used multivariate regression analyses to assess the associations between levels of cotinine and inflammatory markers. RESULTS: The median cotinine level was 0.10 ng/mL (interquartile range, 0.04-0.23 ng/mL). The odds ratios of elevated hs-CRP (above highest CRP tertile, 1.5 mg/L) were 2.85 [95% confidence interval (CI), 1.03-7.89] for the high-cotinine group (> 0.215 ng/mL) and 2.80 (95% CI, 1.11-7.10) for the moderate-cotinine group (0.05-0.215 ng/mL), compared with the low-cotinine group (< 0.05 ng/mL), adjusting for age, sex, race, educational level, obesity, previous smoking history, job title, and medical history. Plasma cotinine levels were not associated with IL-6 or sICAM-1. CONCLUSIONS: SHS exposure, as assessed by plasma cotinine, was positively associated with hs-CRP in this group of blue-collar workers. The strength of the association with hs-CRP depended on the cut points selected for analysis.

Development of an ultrasensitive immunochromatography test to detect nicotine metabolites in oral fluids.

Passive exposure to tobacco smoke causes a variety of illnesses ranging from allergic responses to cancer. Assessment of exposure to second-hand tobacco smoke (SHS), particularly among vulnerable populations enables intervention and prevention of future disease. A minimally invasive oral fluids-based onsite test to detect such exposure would create a valuable tool for researchers and clinicians. Here we describe the development of a test that uses an inexpensive reader that utilizes a CMOS image sensor to reliably quantify a reporter signal and determine nicotine exposure. The rapid lateral flow test consists of a nitrocellulose strip with a control line containing goat anti-rabbit IgG, used as an internal standard, and a test line containing BSA-cotinine conjugate. To run the test, diluted sample containing antibodies against cotinine, the major metabolite of nicotine, is mixed with protein A-gold nanoparticles and placed on the sample pad. As the sample runs up to the nitrocellulose pad, antibodies in the running buffer bind to available cotinine. If cotinine is absent, the antibodies will bind to the BSA-cotinine derivative immobilized on the test line, resulting in an intense purple-red band. The concentration of cotinine equivalents in the sample can be estimated from interpretation of the test line. In this article we describe the effect of different cotinine derivatives, oral fluid pretreatment, and application and running buffers on assay sensitivity. The test can reliably detect as little as 2 ng mL(-1) cotinine equivalents. The assay is sensitive, simple, rapid, inexpensive, and easily implementable in point-of-care facilities to detect second-hand smoke exposure.

Inflammatory markers and secondhand tobacco smoke exposure among U.S. workers.

BACKGROUND: Self-reported exposure to secondhand smoke (SHS) has been associated with elevated inflammatory markers in adults. The association between SHS indicated by serum cotinine and markers of inflammation has not been investigated in adult workers. METHODS: Using the subpopulation of employed participants (20 years and older) who were non-smokers and denied home SHS exposure from the National Health and Nutrition Examination Survey (NHANES) 1999-2002, the association between serum cotinine and inflammatory markers (C-reactive protein, fibrinogen, homocysteine, and white blood cells) was analyzed. Inflammatory marker values were log-transformed and expressed as geometric means with 95% confidence intervals (CI). Serum cotinine was categorized as either no cotinine (below the detection limit), low cotinine (above the detection limit and <0.2 ng/ml), or high cotinine (>or=0.2 and <15.0 ng/ml). The association between serum cotinine and inflammatory markers was analyzed using univariate and multivariate-adjusted linear regression. RESULTS: Geometric mean serum cotinine was significantly higher among non-smokers reporting SHS exposure in the workplace (0.17 vs. 0.10 ng/ml, P < 0.01). Workers exposed to low and high levels of cotinine had significantly higher homocysteine levels relative to non-exposed workers; mean homocysteine differences remained significant in the multivariable model (i.e., 0.363 and 0.491 mg/dl increase, respectively). CONCLUSION: Exposure to SHS as measured by serum cotinine may result in increased homocysteine levels among adult workers. These results provide further evidence in support of universal workplace smoking restrictions in order to protect worker health. Further research is required to determine the adverse effects of workplace SHS exposure on cardiovascular risk.

Development of an anti-cotinine vaccine to potentiate nicotine-based smoking cessation strategies.

Nicotine replacement therapies (NRT) have limited success in smoking cessation. The efficacy of nicotine may be compromised by its main metabolite, cotinine. An anti-cotinine vaccine to remove this antagonism could enhance the efficacy of NRT. We show that cotinine is a weak nicotinic agonist and decreases responses to nicotine, consistent with antagonism through receptor desensitisation. trans-4-Thiol cotinine was coupled to tetanus toxoid, and rats immunised repeatedly. Vaccination raised antibodies specific for cotinine that do not recognise other metabolites or nicotine. Increased serum cotinine concentrations following nicotine administration indicate sequestration of cotinine by antibodies, encouraging further evaluation of this vaccine in behavioural models of nicotine addiction and relapse.

Immunodetection of cotinine protein in granulosa-lutein cells of women exposed to cigarette smoke.

OBJECTIVE: To detect immunoreactivity to cotinine protein, a major metabolite of nicotine, in granulosa-lutein cells from patients exposed to cigarette smoke, as measured by levels of cotinine in follicular fluid (FF) samples. DESIGN: Controlled immunocytochemical study. SETTING: Hospital IVF-ET program treating infertile patients. PATIENT(S): Twenty-eight women classified by self-reported smoking habits: active smokers (n = 17), passive smokers (n = 4), and nonsmokers (n = 7). INTERVENTION(S): Ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Grades of immunostaining intensity were assessed in granulosa-lutein cells. Patient scores of cell immunostaining were calculated and regressed on levels of FF cotinine. RESULT(S): Cotinine levels in FF were higher in active smokers than in passive smokers or nonsmokers. Cotinine immunostaining was visualized in the nucleus and cytoplasm of granulosa-lutein cells. Mean grades and mean scores of immunostaining intensity were higher in active smokers than in passive smokers or nonsmokers. There was a strong positive correlation between scores of cell immunostaining and FF cotinine levels. CONCLUSION(S): The association between cotinine expression in granulosa-lutein cells and FF cotinine provides reliable evidence for a dose-related effect. This constituent of cigarette smoke appears to interact directly with and incorporate into these ovarian cells. Our approach seems useful for monitoring ovarian exposure to environmental toxins.

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.

Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM. A constitutive form of P-450 is also implicated in nicotine metabolism, while purified P-450IA1 and P-450IIC6 show no detectable activity. The lack of P-450IA1 activity substantiates work by others who also failed to observe an increase in the efficiency of nicotine metabolism to cotinine by microsomes from rats that had been pretreated with benzanthracene. This result is in contrast to work with purified rabbit liver enzymes, in which P-450IA1 exhibited low but measurable activity. Our results support the notion that nicotine metabolism to cotinine by P-450 enzymes is highly species dependent. Thus, it is unwise in some cases to extrapolate results obtained by animal model study to the possible role of specific forms of the P-450 enzymes in nicotine metabolism in humans.

Interaction of cotinine with rat hepatic microsomal P-450. Comparison with metyrapone and immunomodulation of cotinine and metyrapone binding by monoclonal anti-cotinine antibodies.

The ability of the major nicotine metabolite, cotinine, to interact with rat liver microsomal cytochrome P-450 and the immunomodulatory effects of anti-cotinine antibodies were studied. Cotinine induced type II spectral changes with both microsomes from phenobarbital (PB)-induced rats and purified P-450 with apparent Ks values of 97 and 750 microM, respectively. In contrast, the Ks value was 0.3 microM for metyrapone and 5 microM for nicotine with both the microsomes and purified enzyme. The apparent Ki value for cotinine inhibition of 7-pentoxyresorufin O-dealkylase activity with the microsomes (87 microM) was approximately 87- and 870-fold higher than for nicotine and metyrapone, respectively. Monoclonal antibodies produced against cotinine cross-reacted equally well with metyrapone. They specifically blocked enzyme binding of both drugs based on dose-dependent inhibition of spectral changes, and reversed the metyrapone-induced inhibition of microsomal O-dealkylase activity. In contrast, antibodies to nicotine did not cross-react with cotinine or metyrapone and had no effect on their activity, although they did block the action of nicotine. These results demonstrate that cotinine binding to P-450 from PB-induced rats and inhibition of functional activity in vitro are qualitatively like the effects of metyrapone and nicotine, and that monoclonal anti-cotinine antibodies are useful molecular probes of the interactions between cotinine and metyrapone with the enzyme.

Idiotype-anti-idiotype hapten immunoassays: assay for cotinine.

Practical application of the idiotype-anti-idiotype reaction to hapten immunoassays has been demonstrated with cotinine as an example. The assay relies on the ability of cotinine, a major nicotine metabolite, to inhibit binding between a monoclonal anti-cotinine antibody (the idiotype) and a second monoclonal antibody (the anti-idiotype) specific for the antigen combining region on the idiotype. A solid phase enzyme-linked immunoadsorbent assay (ELISA) format was adopted in which fluid phase anti-cotinine and cotinine present either as a standard or in a test sample were incubated in microtiter plate wells coated with F(ab')2 fragments of the anti-idiotype. Horseradish peroxidase-labeled protein A and o-phenylenediamine were used to detect idiotype-anti-idiotype binding. Under optimal assay conditions, 0.9 ng cotinine inhibited immune binding by 50% and as little as 0.04 ng could be detected. In contrast, nearly 70 times more trans-3'-hydroxycotinine, a major urinary metabolite, and over 1000-fold more nicotine were required for 50% inhibition. Several other metabolites and structurally related compounds also were poor competitors. Assay reliability was good over a range of cotinine concentrations from 5 to 500 ng/ml saliva with intraassay coefficients of variation between 6 and 10% and interassay values between 6 and 13%. Also, there was a strong correlation (R2 = 0.994) between the cotinine levels found in saliva from 35 cigarette smokers with the idiotype-anti-idiotype assay and a cotinine-anti-cotinine ELISA. Because only monoclonal antibodies and antigen are required, the idiotype-anti-idiotype immunoassay offers a high degree of standardization without the need to prepare labeled hapten derivatives or macromolecular conjugates for solid phase assays.

Comparison of monoclonal and polyclonal antibodies to cotinine in nonisotopic and isotopic immunoassays.

Monoclonal antibodies (McAb) were used to develop nonisotopic and radioimmunoassays (RIA) for quantitative determination of the major nicotine metabolite, cotinine, in physiological fluids. ELISAs and fluorescence immunoassays were carried out in microtiter plate wells coated with a conjugate of cotinine 4'-carboxylic acid bound covalently to poly-L-lysine. The detection systems were horseradish peroxidase (HRP)-labeled staphylococcal protein A, HRP-streptavidin-biotin, and biotinylated alkaline phosphatase-4-methylumbelliferyl phosphate. With the three McAb tested, I50 values ranged between 0.024-0.063 ng cotinine and as little as 0.005-0.015 ng gave 15% inhibition. These assays were 5-20 times more sensitive than similar assays using six rabbit antisera. With McAb the standard inhibition curves were steeper and complete inhibition of immune binding was achieved with approximately 1 ng cotinine. In contrast, 100-500 ng cotinine failed to give greater than 80-90% inhibition with rabbit antibodies either in the plate assays or in RIA using a 125I-labeled tyramine derivative of cotinine as the tracer. In this RIA, the sensitivity with McAb (mean I50 of 0.55 ng cotinine) was over three-fold greater than with rabbit antisera (mean I50 of 1.84 ng). The presence of antibodies directed to the amide linkage group common to the polylysine conjugate. 125I-tyramine derivative and the immunogen likely accounts for the inferior quality of assays using rabbit antisera. Consistent with this conclusion, superimposable inhibition curves were obtained in the RIA when monoclonal or rabbit antibodies were used with [3H]cotinine. Cotinine levels in saliva, serum and plasma from smokers and non-smokers determined with McAb-based assays showed a strong correlation with values obtained by RIA using rabbit antisera or by gas chromatography. Properly selected McAb offer distinct advantages over conventional antisera in nonisotopic immunoassays and RIAs for cotinine as a biochemical marker of active or passive smoking.

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays.

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or continine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10(8) M-1 were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly-L-lysine were coated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5-10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and 3H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) and cotinine (r = 0.981) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.

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.