Development of epitopephore-based rational hapten design strategy: A combination of theoretical evidence and experimental validation.

Antibody is the key biomolecule that governing the sensitivity and specificity of an immunoassay for chemical compound, also named hapten molecule. Obviously, predication of hapten effectiveness before chemical synthesis is beneficial to boost success, save cost and improve controllability. Here, we proposed and evaluated an epitopephore based rational hapten design (ERHD) to assist antibody production to chemical compound, combining theoretical evidence and then experimental validation by using dinitrocarbanilide (DNC) as a model analyte. Briefly, epitopephores of DNC were firstly generated by HipHop algorithm after features mapping. A homemade drug database also containing reported fragment haptens (HFR) and new designed full hapten (HFU) were constructed, and then was virtually screened by using generated epitopephore followed by structural analysis and visual inspection. The DNC haptens based on the selected hits were further identified by Density Functional Theory before total synthesis. To prove and clarify the usability of the ERHD, two retrieved HFU haptens, one non-retrieved HFU hapten and three non-retrieved HFR haptens were all selected to produce monoclonal antibodies (mAbs) for comparison purpose. A maximal 6000-fold increased affinity of mAb from retrieved HFU than HFR was observed, while, non-retrieved HFU failed to produce antibody to DNC. More importantly, mAbs from HFU haptens provided highly specificity to DNC, while, mAbs from HFR haptens could recognize 15 others analogues. We then constructed antibody structure and investigated molecular recognition of the mAbs to DNC, well supporting the rationality of the ERHD. Lastly, an icELISA was developed for DNC with an IC50 value as low as 0.19 ng mL-1 with high specificity, which has never achieved before.

A monoclonal antibody for identifying capsaicin congeners in illegal cooking oil and its applications.

In this work, we studied the preparation of a high-affinity antibody and its immunochromatographic applications to simultaneously identify capsaicin(LJJ), dihydrocapsaicin(HLJ), nordihydrocapsaicin, homodihydrocapsaicin, and other congeners in illegal cooking oil. We used dihydrocapsaicin hapten-conjugated carrier protein BSA as the immunogen according to the formaldehyde method, and conjugated capsaicin and OVA as the coated detection antigen according to the formaldehyde method. We subsequently screened and cloned a hybridoma cell line 2B3 with the highest affinity, which could stably secrete monoclonal antibodies against compounds in the capsaicin family. We then established a capsaicin indirect ELISA standard curve, which was fitted using the linear regression equation R = 0.9987, curve y = -2.3x + 0.2, and IC50 = 0.2 ng/mL. The cross-reaction rate for capsaicin was 100%, 116% for dihydrocapsaicin, 88% for homodihydrocapsaicin, and 94% for nordihydrocapsaicin. In the second application, we established a simple and accurate sample pretreatment method and a quantum dot-labeled test strip to quickly and quantitatively detect capsaicin family compounds in illegal cooking oil in 8 min. The average recovery rates for each spiked concentration were between 75% and 107.8%, and the coefficient of variation values for each spiked concentration were less than 15%. The high-affinity antibody we identified could simultaneously identify capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin, and other congeners in illegal cooking oil, and the antibody could be quickly and accurately applied for the qualitative and quantitative detection of capsaicin family residues in illegal cooking oil.

Contact haptens in emollients marketed in two European countries (Poland and Spain).

INTRODUCTION AND OBJECTIVES: Atopic dermatitis (AD) is the most common skin disease among pediatric patients, which affects up to 20% of children worldwide. Characterized by pruritus and eczema, it is also associated with improper skin barrier function and allergen sensitization. Here, we aimed to assess the presence of haptens in emollients marketed in two European countries: in Poland and Spain, as, firstly, these products are considered to be AD's basic therapy, and, secondly, frequent application of potent sensitizers on atopic skin may result in contact dermatitis. MATERIALS AND METHODS: We systematically searched for moisturizers explicitly described as "Atopic skin care" products in the most frequently visited online pharmacies in Poland and Spain. Subsequently, we created a database of all products and compared their composition with 139 contact haptens listed in the European Baseline Series (EBS), Fragrance and Cosmetic Series. RESULTS: As of December 2018, our list comprised 159 and 111 emollients available on the Polish and Spanish markets, respectively. There were no ingredients listed in 28 (17.5%) products in Poland and 24 (21.6%) in Spain. Only 23 (17.5%) and 13 (14.8%) products were hapten free. The pattern of most common haptens was similar in both countries, including phenoxyethanol, tocopherol and tocopheryl acetate, undefined parfum in Poland and tocopherol, phenoxyethanol, tocopheryl acetate and undefined parfum in Spain. CONCLUSIONS: This study shows that a vast majority of products taken into consideration contain at least one potential contact hapten. These findings indicate a need for patient education about potentially allergenic ingredients and stronger cooperation between academia and cosmetic manufacturers.

Quick and convenient construction of lambda-cyhalothrin antigen for the generation of specific antibody.

Lambda-cyhalothrin is a pyrethroid widely used in crop, fruit and vegetable production, but has potential health threats to human. Immunoassay is a cheap, rapid and facile method to detect lambda-cyhalothrin, yet wide application of this method still requires improvement in the construction of antigen. In this study, we developed a one-step lambda-cyhalothrin hapten synthesis that transformed the cyanide group in lambda-cyhalothrin to amide. Complete antigen was assembled by coupling the amide with succinic-anhydride-activated carrier proteins, and corresponding polyclonal antibodies were generated using Balb/c mice. Using antibody generated by the method in this paper, the competitive ELISA demonstrated the lowest detection limit of 3.772 µg/L for lambda-cyhalothrin, and no significant cross-reactivity for other pyrethroid pesticides was observed. All the results suggested we have established a more efficient technique of generating lambda-cyhalothrin antibody. Furthermore, since the activated proteins used in this study are highly controllable, we believe these proteins could potentially be the prototype of a series of standardized carrier proteins for the synthesis of complete antigens.

Design of Novel Haptens and Development of Monoclonal Antibody-Based Immunoassays for the Simultaneous Detection of Tylosin and Tilmicosin in Milk and Water Samples.

In this work, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established to detect tylosin and tilmicosin in milk and water samples. A sensitive and specific monoclonal antibody was prepared by rational designed hapten, which was achieved by directly oxidizing the aldehyde group on the side chain of tylosin to the carboxyl group. Under the optimized conditions, the linear range of icELISA for tylosin and tilmicosin were 1.3 to 17.7 ng/mL and 2.0 to 47.4 ng/mL, with half-maximal inhibition concentration (IC50) values of 4.7 and 9.6 ng/mL, respectively. The cross-reactivity with other analogues of icELISA was less than 0.1%. The average recoveries of icELISA for tylosin and tilmicosin ranged from 76.4% to 109.5% in milk and water samples. Besides, the detection results of icELISA showed good correlations with HPLC-MS/MS. The proposed icELISA was satisfied for rapid and specific screening of tylosin and tilmicosin residues in milk and water samples.

Development of an on-line analytical platform to screen haptens in shuxuening injection based on high-performance liquid chromatography coupled with ion-trap multistage mass spectrometry and human serum albumin fluorescence detection.

Shuxuening injection (SXNI), one of the traditional Chinese medicine injections (TCMI), is widely used for the treatment of cardiovascular diseases in the clinic. However, its allergic reactions have impeded the clinical applications of SXNI, such adverse reactions have not been well understood due to the lack of methods for detecting haptens. In this study, a high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-human serum albumin-fluorescence detector (HPLC-DAD-MSn-HSA-FLD) system was established to identify and screen haptens for the first time. Flavones, flavonols and their glycosides in SXNI showed strong HSA binding ability in different degrees. Fifteen of these compounds were used to study the association of HSA binding ability and sensitizability using isothermal titration calorimetry (ITC) and fluorescence techniques, furthermore, RBL-2H3 cell experiments were conducted to verify the results. It was found that ginkgolides showed no sensitizability, while flavones and flavonol aglycones showed stronger sensitizability than their glycosides. The system was proven to be precise, stable and reproducible, which lays a foundation for screening haptens in SXNI and relevant samples.

Engineered magnetosomes fused to functional molecule (protein A) provide a highly effective alternative to commercial immunomagnetic beads.

BACKGROUND: Magnetosomes (also called bacterial magnetic nanoparticles; BMPs) are biomembrane-coated nanoparticles synthesized by magnetotactic bacteria (MTB). Engineered BMPs fused to protein A (termed ∆F-BMP-FA) bind antibodies (Abs) automatically, and thus provide a series of potential advantages. However, no report so far has systematically evaluated functional applicability of genetically engineered BMPs. RESULTS: We evaluated properties of ∆F-BMP-FA, and developed/optimized culture methods for host strain Magnetospirillum gryphiswaldense ΔF-FA, ∆F-BMP-FA extraction conditions, conditions for Ab conjugation to ∆F-BMP-FA surface, and procedures for antigen detection using ∆F-BMP-FA/Ab complexes (termed BMP-A-Ab). Fed-batch culture for 36 h in a 42-L fermentor resulted in yields (dry weight) of 2.26 g/L for strain ΔF-FA and 62 mg/L for ∆F-BMP-FA. Optimal wash cycle number for ∆F-BMP-FA purification was seven, with magnetic separation following each ultrasonication step. Fusion of protein A to BMPs resulted in ordered arrangement of Abs on BMP surface. Linkage rate 962 µg Ab per mg ∆F-BMP-FA was achieved. BMP-A-Ab were tested for detection of pathogen (Vibrio parahaemolyticus; Vp) surface antigen and hapten (gentamicin sulfate). Maximal Vp capture rate for BMP-A-Ab was 90% (higher than rate for commercial immunomagnetic beads), and detection sensitivity was 5 CFU/mL. ∆F-BMP-FA also bound Abs from crude mouse ascites to form complex. Lowest gentamicin sulfate detection line for BMP-A-Ab was 0.01 ng/mL, 400-fold lower than that for double Ab sandwich ELISA, and gentamicin sulfate recovery rate for BMP-A-Ab was 93.2%. CONCLUSION: Our findings indicate that engineered BMPs such as ∆F-BMP-FA are inexpensive, eco-friendly alternatives to commercial immunomagnetic beads for detection or diagnostic immunoassays, and have high Ab-conjugation and antigen-adsorption capacity.

Sensitivity of KeratinoSensTM and h-CLAT for detecting minute amounts of sensitizers to evaluate botanical extract.

Cosmetic ingredients are often complex mixtures from natural sources such as botanical extracts that might contain minute amounts of constituents with sensitizing potential. The sensitivity of in vitro skin sensitization test methods such as KeratinoSensTM and h-CLAT for the detection of minute amounts of sensitizer in mixtures remains unclear. In this study, we assessed the detection sensitivity of the binary test battery comprising KeratinoSensTM and h-CLAT for minute amounts of sensitizers by comparing the LLNA EC3 (estimated concentration of a substance expected to produce a stimulation index of 3) values to the minimum detection concentrations (MDCs) exceeding the positive criteria for each of the two in vitro test methods. 146 sensitizers with both sets of in vitro data and LLNA data were used. MDC values for KeratinoSensTM and h-CLAT were calculated from exposure concentrations exceeding positive criteria for each in vitro test method (EC1.5 and minimum induction thresholds, respectively). The dilution rate used to expose culture medium was also considered. For 86% of analyzed sensitizers, the in vitro test methods showed MDC values lower than LLNA EC3 values, suggesting that the binary test battery with KeratinoSensTM and h-CLAT have greater sensitivity for detection of minute amounts of sensitizer than LLNA. These results suggest the high applicability of KeratinoSensTM and h-CLAT for detecting skin sensitizing constituents present in botanical extract.

A label-free diffraction-based sensing displacement immunosensor to quantify low molecular weight organic compounds.

Herein we present a diffractometric immunosensor to quantify low molecular weight organic compounds in a label-free, simple, and sensitive fashion. The approach is based on patterning analyte analogues (haptens) on solid surfaces according to a diffractive structure, and then loading specific antibodies on them to be subsequently displaced by free analytes in solution. This displacement generates a measurable change in the diffractive response that enables to quantify the analyte concentration. In this study we address the fabrication, optimization, and assessment of these diffractive structures of biological probes and their application to the analysis of atrazine, an organic compound extensively used as pesticide. This immunosensor displays well-correlated dose-response curves that reach a detection limit of 1.1 ng mL-1 of atrazine in label-free conditions. From a general viewpoint, this study also aims to provide insights into exploiting this approach towards prospective in-field analysis and screening strategies to sense multiple low molecular weight compounds in label-free conditions.

Effects of Gamma and Electron Radiation on the Structural Integrity of Organic Molecules and Macromolecular Biomarkers Measured by Microarray Immunoassays and Their Astrobiological Implications.

High-energy ionizing radiation in the form of solar energetic particles and galactic cosmic rays is pervasive on the surface of planetary bodies with thin atmospheres or in space facilities for humans, and it may seriously affect the chemistry and the structure of organic and biological material. We used fluorescent microarray immunoassays to assess how different doses of electron and gamma radiations affect the stability of target compounds such as biological polymers and small molecules (haptens) conjugated to large proteins. The radiation effect was monitored by measuring the loss in the immunoidentification of the target due to an impaired ability of the antibodies for binding their corresponding irradiated and damaged epitopes (the part of the target molecule to which antibodies bind). Exposure to electron radiation alone was more damaging at low doses (1 kGy) than exposure to gamma radiation alone, but this effect was reversed at the highest radiation dose (500 kGy). Differences in the dose-effect immunoidentification patterns suggested that the amount (dose) and not the type of radiation was the main factor for the cumulative damage on the majority of the assayed molecules. Molecules irradiated with both types of radiation showed a response similar to that of the individual treatments at increasing radiation doses, although the pattern obtained with electrons only was the most similar. The calculated radiolysis constant did not show a unique pattern; it rather suggested a different behavior perhaps associated with the unique structure of each molecule. Although not strictly comparable with extraterrestrial conditions because the irradiations were performed under air and at room temperature, our results may contribute to understanding the effects of ionizing radiation on complex molecules and the search for biomarkers through bioaffinity-based systems in planetary exploration.

Fluxapyroxad Haptens and Antibodies for Highly Sensitive Immunoanalysis of Food Samples.

Fluxapyroxad is a new-generation carboxamide fungicide, with residues increasingly being found in food samples. Immunochemical assays have gained acceptance in food quality control as rapid, cost-effective, sensitive, and selective methods for large sample throughput and in situ applications. In the present study, immunoreagents to fluxapyroxad were obtained for the first time, and competitive immunoassays were developed for the sensitive and specific determination of fluxapyroxad residues in food samples. Two carboxyl-functionalized analogues of fluxapyroxad were prepared, and antibodies with IC50 values in the low nanomolar range were generated from both haptens, though a dissimilar response was observed concerning specificity. A robust direct assay was set up, with a calibration curve exhibiting a limit of detection of 0.05 nM (0.02 µg/L). Limits of quantitation of 5 µg/L were obtained for peach, apple, and grape juices using samples diluted in water. The direct immunoassay was also successfully applied to the determination of fluxapyroxad in grapes from in-field treated grapevines.

The Preparation and Identification of a Monoclonal Antibody against Domoic Acid and Establishment of Detection by Indirect Competitive ELISA.

Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 × 108 L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006-0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56% ± 2.8% with the coefficient variation of 0.01-0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.

An on-line analytical approach for detecting haptens in Shuang-huang-lian powder injection.

Shuang-huang-lian powder injection (SHLPI) is a traditional Chinese medicine injection (TCMI) frequently used in the clinical treatment of faucitis, bronchitis, and other viral and bacterial infections of upper respiratory tract. However, its allergenic reactions, being the main adverse effects (AEs) of SHLPI, have been a serious problem of its clinical safety. This problem has not been solved due to short of methods for detecting haptens in complex TCMIs. In this study, an on-line high-performance liquid chromatography-diode-array detector-mass spectrometry combined with bovine serum albumin-fluorescence detector (HPLC-DAD-MS-BSA-FLD) system was established for the first time, validated and applied for identification of haptens in SHLPI. Fourteen of 35 identified compounds showed BSA binding activity, and they were six flavonoids, six caffeoylquinic acids (CQAs), and two phenylethanoid glycosides. The structure-activity relationships of 10 active components were studied, and their ability of sensitization together with that of two CQAs were further verified by ELISA assay. It was found that 10 compounds had sensitization, and flavonoids showed stronger sensitizability than CQAs while the diCQAs were slightly stronger than caffeoylquinic acids. The system was validated using 3-CQA as a positive control, and was proved to have good reproducibility, stability, precision (RSD<0.1%) and linearity (R2>0.9993). This online system is fast, sensitive and efficient for screening haptens in traditional Chinese medicine injection (TCMI), provides a new approach to reveal the chemical basis of haptens in TCMIs.

Detection of okadaic acid (OA) using ELISA and colloidal gold immunoassay based on monoclonal antibody.

Okadaic Acid (OA), a small seafood-borne toxin secreted by Dinophysis and Prorocentrum dinoflagellates, is generally distributed in various species of shellfish and has caused diarrhetic shellfish poisoning (DSP). In view of OA toxin threat to humans and animals, it is essential to develop a rapid, accurate and sensitive method for the detection and quantification of OA in real samples. In this study, a monoclonal antibody named 10E8 was screened by cells fusion of Sp2/0 with spleen cells isolated from immunized mouse, and the isotype of McAb 10E8 was belonged to IgG1. The resulted McAb 10E8 displayed higher specificity to OA antigen, with the highest affinity of 2.66×109L/moL until now. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect OA was 20-750ng/mL. The limit of detection (LOD) was 12pg/mL, and the recovery average was (84.04±5.08)%. The LOD of colloidal gold immunoassay by naked eye and strip reader was 1ng/mL and 100pg/mL, respectively, with an average recovery of (88.0275±4.4225)%. Therefore, the developed ELISA and colloidal gold immunoassay based on this McAb can be used for OA detection in real samples.

A sensitive chemiluminescent immunoassay to detect Chromotrope FB (Chr FB) in foods.

Chromotrope FB (Chr FB) is a synthetic azo dye permitted for use in foods and medicines. An acceptable daily intake (ADI) of Chr FB was 0-0.5mg/kg in China. In this study, we synthesized a Chr FB hapten with an amino group to prepare its artificial immunogen. Polyclonal antibodies obtained from New Zealand rabbits were applied to develop an indirect competitive chemiluminescent immunoassay (icCLIA) to detect Chr FB in foods. A horseradish peroxidase (HRP)-luminol-H2O2 system was used to yield CL signal with p-iodophenol as an enhancement reagent. The method showed good specificity towards Chr FB and could detect as low as 0.02ngmL-1 Chr FB in buffer, 0.07ngg-1 in yoghurt candy, 0.07ngg-1 in vitamin drink and 0.13ngg-1 in bread. Compared with HPLC method, the proposed method is more sensitive by two orders of magnitude. The accuracy and precision of this method are acceptable and comparable with HPLC method. Therefore, the proposed method could be used for rapid screening of Chr FB in the mentioned foodstuffs.

Development of an enzyme linked immunosorbent assay for the determination of phenothiazine drugs in meat and animal feeds.

In this study, 2-chlorophenothiazine was used to synthesize a hapten for production of monoclonal antibody. The obtained monoclonal antibody showed high crossreactivities to chlorpromazine, promethazine and perphenazine, and showed low crossreactivities to acepromazine and fluphenazine. After evaluation of three coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the five phenothiazines in animal feeds and the residues of chlorpromazine, promethazine and perphenazine in meat. The crossreactivities to the five analytes were in a range of 2.4%-98%. The limits of detection for the five drugs in feeds were in a range of 0.1-3.0 µg g-1, and that for chlorpromazine, promethazine and perphenazine in meat were in a range of 0.5-0.8 ng g-1. Their recoveries from standards fortified blank samples (chicken, pork and feeds) were in a range of 74.1%-96.5% with coefficients of variation of 6.4%-15.1%. Therefore, this method could be used as a rapid screen tool to determine phenothiazine drugs in animal feeds and animal derived foods.

Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay.

In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL(-1) and 19.97 ± 0.84 ng mL(-1), respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of "nanobody (N-28) - anti-DON MAb" complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 - Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants.

Selection of bisphenol A - single-chain antibodies from a non-immunized mouse library by ribosome display.

Developing reagents with high affinity and specificity are critical to detect the environmental hormones or toxicants. Ribosome display technology has been widely used in functional protein or peptide screening and in directed evolution of protein molecules in vitro. In this study, single-chain variable fragments (scFvs) against bisphenol A (BPA) were selected from a library constructed from splenocytes of non-immunized mice. After five rounds of selection, the selected scFvs bound to BPA with high affinity. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was introduced to screen the antibody affinity and specificity to BPA. The equilibrium dissociation constants (KDS) of one clone was 1.76µM as determined by surface plasmon resonance (SPR). This study indicated that ribosome display can isolate binders to small molecules from a non-immunized naive library without any in vivo steps and can generate recombinant antibodies efficiently and rapidly. In addition, this study provides a methodological framework for detection of small molecules using recombinant antibodies.

Simple radiometric method for accurately quantitating epitope densities of hapten-protein conjugates with sulfhydryl linkages.

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses (14)C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten-BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (r(2) = 0.98, y = 0.98x + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten-protein conjugations, and choosing the most effective epitope densities for conjugate vaccines.

Implants and contact allergy: are sensitizing metals released as haptens from coronary stents?

BACKGROUND: The possible impact of metal release from coronary artery stents has, with their increased use, become a concern. OBJECTIVES: To study in vitro metal release in biologically relevant milieu from coronary stents made of different alloys. MATERIALS AND METHOD: Coronary stents in common use in a department of cardiology at the time of the study were tested. A previously described in vitro technique was used, whereby the stents were kept in the extraction media for a week. Two different extraction media were used to show the necessity of studying the actual biological surrounding of the implant when metal release is investigated. Metal release was determined with atomic absorption spectrometry. RESULTS: In this study, we show metal release from stents after immersion in extraction media of artificial sweat and cysteine solution, as illustrative media. CONCLUSION: Metal release from coronary stents is shown. The magnitude of release is influenced by several factors. The extent to which metal release in vitro has potential biological effects, in terms of elicitation of an allergic reaction or induction of sensitization, in vivo needs to be explored. However, as metal release from an implant in a biologically appropriate medium has been established, better risk assessments in relation to delayed hypersensitivity may be undertaken.