Rationalization of a nanoparticle-based nicotine nanovaccine as an effective next-generation nicotine vaccine: A focus on hapten localization.

A lipid-polymeric hybrid nanoparticle-based next-generation nicotine nanovaccine was rationalized in this study to combat nicotine addiction. A series of nanovaccines, which had nicotine-haptens localized on carrier protein (LPKN), nanoparticle surface (LPNK), or both (LPNKN), were designed to study the impact of hapten localization on their immunological efficacy. All three nanovaccines were efficiently taken up and processed by dendritic cells. LPNKN induced a significantly higher immunogenicity against nicotine and a significantly lower anti-carrier protein antibody level compared to LPKN and LPNK. Meanwhile, it was found that the anti-nicotine antibodies elicited by LPKN and LPNKN bind nicotine stronger than those elicited by LPKN, and LPNK and LPNKN resulted in a more balanced Th1-Th2 immunity than LPKN. Moreover, LPNKN exhibited the best ability to block nicotine from entering the brain of mice. Collectively, the results demonstrated that the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine could be enhanced by modulating hapten localization, providing a promising strategy to combatting nicotine addiction.

An Advance in Prescription Opioid Vaccines: Overdose Mortality Reduction and Extraordinary Alteration of Drug Half-Life.

Prescription opioids (POs) such as oxycodone and hydrocodone are highly effective medications for pain management, yet they also present a substantial risk for abuse and addiction. The consumption of POs has been escalating worldwide, resulting in tens of thousands of deaths due to overdose each year. Pharmacokinetic strategies based upon vaccination present an attractive avenue to suppress PO abuse. Herein, the preparation of two active PO vaccines is described that were found to elicit high-affinity antiopioid antibodies through a structurally congruent drug-hapten design. Administration of these vaccines resulted in a significant blockade of opioid analgesic activity, along with an unprecedented increase in drug serum half-life and protection against lethal overdose.

Noncompetitive immunoassay detection system for haptens on the basis of antimetatype antibodies.

BACKGROUND: Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies. METHODS: We generated antimetatype monoclonal antibodies against a hapten-antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated. RESULTS: The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%-12.6% (intraassay) and 6.2%-21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%-2.3% (intraassay) and 1.9%-3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available (125)I RIA. CONCLUSIONS: Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.

Unusual antibiotic presence in gym trained subjects with food intolerance; a case report / Presencia de antibióticos inusual en el gimnasio entrenado sujetos con intolerancia a los alimentos; informe de un caso; estudio preliminar

Introduction: Great interest is raising in food intolerances due to the lack, in many cases, of a particular sensitizing agent. Objective: We investigated the serum level of possible new haptens in 15 heavy meat consumers for sport fitness affected by various kinds of food intolerance and who had ever been administered antibiotics in their life for clinical problems. Methods: Forty ml of blood were drawn from each patient and analyzed, by means of an ELISA test, in order to possibly identify the presence of an undue contaminant with hapten properties. Results: Four out of fifteen subjects (26%) showed a serum oxytetracycline amount > 6 ng/g (which is considered the safety limit), 10 of 15 (66%) a serum doxycycline amount > of 6 ng/g and 3 out of 15 (30%) subjects had high serum level of both molecules. Conclusions: Although a direct ratio between body antibiotics remnant storage in the long run and chronic gut dysfunctions and/or food allergy did not reached the evidence yet, the blood traces of these compounds in a food intolerant otherwise healthy population might be considered the preliminary putative step of a sensitizing pathway. Our next goals foresee a deeper insight into the sensitizing trigger from human chronic antibiotic exposure via the zootechnical delivery of poultry food (AU)

Introducción: La falta, en muchos casos, de un agente sensibilizador está despertando un enorme interés en la tolerancia a los alimentos. Objetivo: Investigamos el nivel sérico de posibles nuevos haptenos en 15 grandes consumidores de carne para entrenamiento deportivo afectados por diversos tipos de intolerancia a los alimentos y que habían recibido antibióticos en algún momento de sus vidas por problemas médicos. Métodos: Se realizaron extracciones de sangre de 40ml a cada paciente y se analizaron empleando un test ELISA, para identificar la posible presencia de un elemento contaminante indebido con propiedades de hapteno. Resultados: 4 de 15 sujetos (26%) mostraron una cantidad de oxitetraciclina en suero > 6 ng/g (considerado el límite de seguridad); 10 de 15 (66%) sujetos presentaron una cantidad de doxiciclina > 6 ng/g; y 3 de 15 (30%) sujetos presentaron un alto nivel sérico de ambas moléculas. Conclusiones: Aunque no se llegó a obtener evidencia de una relación directa entre la acumulación de antibióticos corporales a largo plazo y una disfunción intestinal crónica y/o alergias a los alimentos, las trazas en sangre de estos compuestos en una población con alguna intolerancia a los alimentos pero por lo demás sana, podría considerarse el primer paso de una vía de sensibilización. Nuestros próximos objetivos prevén un estudio más profundo del desencadenante sensibilizador a partir de la exposición crónica a antibióticos en humanos por medio de la administración zootécnica de comida avícola (AU)

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis.

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.

Role of intestinal Bifidobacterium pseudolongum in dietary fructo-oligosaccharide inhibition of 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice.

Strategies to manipulate the gut microbiota have been explored for preventing allergy development. We previously showed that dietary supplementation with fructo-oligosaccharide (FOS) reduced 2, 4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS) in BALB/c mice. Because the CHS response was negatively correlated with the number of faecal bifidobacteria, particularly Bifidobacterium pseudolongum, the present study aimed to examine whether oral administration of B. pseudolongum affects CHS response. Viable B. pseudolongum was successfully isolated from mouse faeces. Female BALB/c mice were fed a synthetic diet with or without FOS supplementation, and B. pseudolongum (2 x 10(7) cells) was administered daily throughout the experimental period. Two weeks after starting the test diets, mice received DNFB on the ear auricle twice at 7-d intervals. Conventional cultivation and molecular biological analyses based on 16S rRNA gene sequences showed that administration of FOS and B. pseudolongum resulted in higher excretion of viable bifidobacteria, mainly B. pseudolongum. Although dietary FOS reduced the CHS response as demonstrated by ear swelling, B. pseudolongum administration resulted in a reduction in the initial phase only of the CHS response. B. pseudolongum administration increased hapten-specific IgG1, while dietary FOS decreased IgG2a in sera. Administration of FOS and B. pseudolongum decreased interferon-gamma production and increased IL-10 production in cervical lymph node cells restimulated with hapten in vitro. We conclude that B. pseudolongum proliferation in the intestinal tract is partially responsible for the reduction in DNFB-induced CHS response by dietary supplementation with FOS in mice, which may be mediated by the modulation of antigen-induced cytokine production.

Use of biotin-streptavidin system for developing a viable, sensitive and specific antigen heterologous assay for hapten.

The present study demonstrates improvement in sensitivity and specificity of hapten assay by using antigen heterology in conjunction with low molecular weight biotin label as compared to high molecular weight horseradish peroxidase (HRP) label. For generation of antiserum, cortisol-3-O-carboxylmethyl-oxime-bovine serum albumin (F-3-CMO-BSA) was used as immunogen whereas, for the preparation of primary label, corticosterone-3-carboxymethyl oxime (B-3-CMO) was coupled with biotinylcaproylhydrazide and HRP by employing N-hydroxysuccinimide mediated carbodiimide reaction. The data of the present study revealed that the antigen heterologous assay which employed high molecular weight HRP label showed 100% cross-reaction with corticosterone. On the contrary, when HRP was replaced with low molecular weight biotin label, less than 0.1% cross-reaction was observed with all analogous C(18), C(19), C(21) and C(27) steroids including corticosterone (0.2%). Moreover, the sensitivity of the later assay was 0.09 microg/dL, which is appreciable as compared to previously reported enzyme based assays. The recovery of the exogenously spiked serum pools lies in the range of 90.3-104.2%. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 3.3% to 7.8% and 2.3% to 7.7%, respectively. The serum cortisol values obtained by this method correlated well with those obtained by radioimmunoassay; r=0.9 (n=50). The use of much stable biotin label in place of HRP has made the antigen heterologous enzyme linked immunosorbent assay (ELISA) of cortisol assay highly specific and sensitive.

Generating recombinant anti-idiotypic antibodies for the detection of haptens in solution.

A new method is described for generating recombinant human and chicken antibody fragments for accurate quantification of haptens in solution. The chemistry of labelling small molecules has always been a problem in the development of immunoassays. Here, we describe a specific panning procedure that enables the selection of recombinant anti-idiotypic phage antibodies that bind to hapten binding molecules (e.g., antibodies) in the absence of the hapten, but are displaced in a highly specific and concentration dependent manner, in the presence of the hapten. The major advantage of such a detection system is that there is no need to label the hapten or to covalently attach it to a solid phase. In this study we demonstrate, using cortisol and aldosterone as model haptens, that the recombinant antibody phage display technology offers great possibilities to generate recombinant anti-idiotypic antibodies. Furthermore, we show that such antibodies can be used successfully to design highly sensitive immunoassays for the quantification of small molecules.

Analytical performance of luminescent immunoassays of different format for serum daidzein analysis.

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/H2O2/enhancer). The second immunoassay is based on the use of bovine serum albumin-daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.

Performance of competitive and indirect enzyme-linked immunosorbent assays, gel immunoprecipitation with native hapten polysaccharide, and standard serological tests in diagnosis of sheep brucellosis.

Competitive and standard enzyme-linked immunosorbent assays (ELISAs), rose bengal (RB), complement fixation, and agar gel immunoprecipitation with native hapten (AGID-NH) were compared by using sera from Brucella-free, Brucella melitensis-infected, and B. melitensis Rev1-vaccinated sheep. The most sensitive tests were indirect ELISA and RB, and the most specific tests were AGID-NH and competitive ELISA. We show that RB followed by AGID-NH is a simple and effective system for diagnosing sheep brucellosis.

Pharmacokinetic analysis of the perivascular distribution of bifunctional antibodies and haptens: comparison with experimental data.

A mathematical model is developed to describe the concentration profiles around individual tumor blood vessels for two-step approaches to cancer treatment. The model incorporates plasma pharmacokinetics, interstitial diffusion, reversible binding between antibody and hapten and between antibody and tumor-associated antigens, and physiological parameters to evaluate present experimental approaches and to suggest new guidelines for the effective use of two-step approaches. Results show considerable interaction between the binding kinetics, initial drug doses, and antigen density, with optimal parameter ranges depending on the desired goal: treatment or detection. The hapten concentration in tumors was found to be nonuniform because of specific binding to antibodies. While binding of the hapten to the bifunctional antibody is necessary for improved retention, too large a binding affinity may lead to very poor penetration of the hapten into regions far away from blood vessels. The time delay between antibody and hapten injection was found to be an important parameter. Longer time delays were found to be advantageous, subject to constraints such as internalization of the antibody and tumor growth during treatment. A proper combination of initial doses for the two species was also seen to be crucial for maximum effectiveness. Comparison of the model with the experimental data of Le Doussal et al. (Cancer Res., 51: 6650-6655, 1991) and Stickney et al. (Cancer Res., 50: 3445-3452, 1990) suggests two novel, yet testable, hypotheses: (a) the early pharmacokinetics of low molecular weight agents can have an important effect on later concentrations using two-step approaches; and (b) metabolism may play an important role in reducing concentrations in the tumor and tumor:plasma concentration ratios. These results should help in the effective design of two-step strategies.

Fast filtration enzyme immunoassay for haptens.

An immunometric method for determination of hapten concentration in fluids has been developed. High-affinity hapten-specific enzyme-labeled monoclonal antibodies are mixed with a sample containing hapten, then the mixture is filtered through a membrane with immobilized hapten. The level of enzyme activity retained by the membrane is inversely proportional to the concentration of hapten in a sample. The assay has been developed for theophylline, digoxin and phenobarbital. The coefficient of variation is less than 5% and the test takes about 2 min.

Multilayer fluorescent immunoassay technique.

We describe a new multilayer immunoassay element for the determination of haptens in undiluted serum and plasma. Polysaccharide layers are coated onto a plastic base. The signal layer contains an immobilized antibody and a fluorescent-labeled hapten. A second layer, containing a pigment, acts as an optical screen. Sample spreading is achieved by a molded grid in contact with the upper layer of the immunoassay element. After sample is added to the element, endogenous analyte competes with the labeled hapten for the binding sites of the immobilized antibody; equilibrium is reached in 4-12 min. Because of the relative liquid-holding capacities of the layers and the grid, only a small amount of the free components remains in the signal layer. The signal is measured by front-surface fluorimetry. This technology has been applied to theophylline and thyroxin assays. Within- and between-run CVs range from 3% to 6%. Comparisons with fluorescent polarization immunoassays (Abbott TDx) showed excellent correlation (theophylline: r = 0.98, slope = 1.07, intercept = 0.3; thyroxin: r = 0.97, slope = 0.91, intercept = 0.8). The new method requires only one pipetting step (sample delivery) and is potentially applicable to a wide range of analytes.