Syntheses of haptens and hapten-protein conjugates for insecticide propoxur and cyhalothrin

A facile procedure was described for the hapten design of the N-methylcarbamate insecticide propoxur.Two new haptens of propoxur(hapten 1a and hapten 1b) were synthesized by introducing appropriate spacers in the pesticide aromatic moiety of the analyte molecular structure.First,the propoxur reacted with nitric acid to yield the intermediate product.Then hapten 1a was prepared via the reduction of the intermediate product,and hapten 1b was formed by the acylation of hapten 1a with succinic anhydride.In addi...     

Design and synthesis of an alpha,alpha-difluorophosphinate hapten for antibody-catalyzed hydrolysis of organophosphorus nerve agents

In a new approach to the safe neutralization of organophosphorus chemical weapons, we designed a hapten to elicit catalytic antibodies with phosphatase activity. Here we report the synthesis of this alpha,alpha-difluorophosphinate hapten 6. Various methods for the introduction of the key alpha,alpha-difluoromethyl feature into the phosphinate hapten are discussed. The best results were obtained with the electrophilic gem-difluorinating agent N-fluorobenzenesulfonimide.     

Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion

Five haptens with different spacer-arm attachment sites on the structure of the organophosphorus insecticide fenthion were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and three haptens containing all or most of the structure of fenthion were conjugated with bovine serum albumin (BSA) for the immunogen. Six polyclonal antisera were raised against the three BSA conjugates, and 30 antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for fenthion. The study revealed the best combination with high sensitivity (I₅₀ of 0.08 ng mL⁻¹) and high assay specificity, which indicated that when structural difference between the analyte and an immunizing hapten is less than that between a coating hapten and the immunizing hapten, a high sensitive enzyme-linked immunosorbent assay (ELISA) in the heterologous system may stand a good chance to be developed. The immunity results showed that heterology in the hapten spacer-arm attachment site of the immunogen could achieve a remarkable improvement in the quantity, sensitivity, and/or specificity of antibody, and that the moiety of an analyte, which is the same as the moiety near/on the immunizing spacer-arm hapten attachment site, contributes greatly to the interaction of antibody and hapten.     

半抗原的设计、修饰及人工抗原的制备

制备具有良好免疫原性的人工抗原是建立测定小分子化合物的免疫分析方法的最关键步骤.本文对半抗原的设计、修饰(修饰位点的选择、修饰物的制备、连接臂的选择)、载体的选择、半抗原与载体的偶联(偶联方式、偶联条件、最佳偶联率)、人工抗原的纯化与鉴定等方面作了详细的介绍.     

去甲氯胺酮半抗原及其全抗原的合成与鉴定

在低温条件下,去甲氯胺酮与琥珀醛酸反应,合成了半抗原羧基-去甲氯胺酮,电喷雾质谱鉴定结果表明,目标半抗原合成成功;通过碳二亚胺法将半抗原与载体蛋白偶联制备人工抗原,红外光谱法鉴定结果表明,人工抗原合成成功,基质辅助激光解析电离飞行时间质谱鉴定表明去甲氯胺酮半抗原与牛血清白蛋白的偶联比为11:1。经动物免疫,获得高效价特异性多克隆抗体,抗血清效价可达5.12×104。     

新型游离三碘甲腺原氨酸人工抗原的合成与表征

利用快速、温和的“Click Chemistry(点击化学)”反应,在游离三碘甲腺原氨酸(FT3)的羧基上引入一个带有三唑环的羧基“间接臂”,生成半抗原(FT3C)。 再用N-羟基琥珀酰亚胺活性酯法,将半抗原(FT3C)分别与牛血清白蛋白(BSA)和钥孔戚血蓝素(KLH)偶联,合成了免疫抗原(FT3C-BSA) 和包被抗原(FT3C-KLH)。 免疫抗原(FT3C-BSA)的紫外吸收光谱相对于FT3C和BSA有明显差异,表明成功实现了偶联,经计算得到偶联比为7∶1;用FT3C-BSA免疫Balb/c小鼠,获得高效价的抗FT3血清。 采用间接ELISA法检测抗体的IC50值为0.37 mg/L,抗血清效价可达6.24×104,是制备FT3人工抗原的一种新方法。     

环境荷尔蒙类物质2,4,6-三氯酚人工抗原的合成

以环境荷尔蒙类物质2,4,6-三氯酚(TCP),氯乙酸为起始原料,经2步反应合成了2,4,6-三氯苯氧乙酸(1)。通过混合酸酐法将1分别与载体蛋白质BSA,OVA偶联获得了TCP的免疫抗原(TCP-BSA)和包被抗原(TCP-OVA)。TCP-BSA免疫兔获得了具有较高效价的抗TCP的多克隆抗体。     

硫代磷酸二乙酯类农药半抗原设计及抗体识别特性

通过分析硫代磷酸二乙酯类农药的结构特点, 设计并合成了系列半抗原; 采用活泼酯法将半抗原分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联制备了系列免疫原和包被原; 通过免疫新西兰大白兔获得了相应抗硫代磷酸二乙酯类农药的类特异性抗体. 建立检测硫代磷酸二乙酯类农药的间接竞争酶联免疫分析(ELISA)方法, 分析探讨了免疫半抗原结构对抗体特性的影响, 并阐述了包被半抗原结构对ELISA灵敏度的影响规律. 结果表明, 手臂取代位置在苯环对位且手臂较短的免疫原具有较好的免疫效果, 同时异源包被可以显著提高ELISA方法的灵敏度. 由抗体PAb-H1和包被原H6-OVA建立的间接竞争ELISA方法可以同时检测7个广泛使用的有机磷农药, 其半抑制浓度(IC50)分别为蝇毒磷(0.013 mg/L)、对硫磷(0.348 mg/L)、喹硫磷(0.022 mg/L)、三唑磷(0.035 mg/L)、甲拌磷(0.751 mg/L)、除线磷(0.850 mg/L)及辛硫磷(1.301 mg/L), 最低检测限符合国内外相关有机磷药物最大允许残留限量标准(MRLS)的检测要求.     

Determination of β-adrenergic agonists by hapten microarray

The use of highly active β-agonists as growth promoters is not appropriate because of the potential hazard for human and animal health. To investigate the residue level of these β-agonists, hapten microarrays were employed for clenbuterol (CLB), ractopamine (RAC) and salbutamol (SAL) residue analysis. CLB, RAC and SAL conjugates were immobilized on the slides, which were precoated by agarose film to construct hapten microarrays, and then the corresponding monoclonal antibodies of these β-agonists and the standards or samples were introduced for indirect competitive immunoassay. Finally, Cy3-labeled secondary antibody was employed to indicate the antigen-antibody complex. The fluorescence intensity of each spot was imaged and recorded, and the calibration curve of each analyte was obtained by plot fluorescence intensity against different standard concentrations. Compared to the ELISA, the hapten microarray method was more sensitive, which got the detection limits 0.09 μg/L for CLB, 0.50 μg/L for RAC, and 0.01 μg/L for SAL. What's more, with the recovery rate between 96.5% and 106.4%, and the coefficient of variation below 10%, the proposed hapten microarray method was shown to be both quantitative and reproducible.     

Broadening the Aldolase Catalytic Antibody Repertoire by Combining Reactive Immunization and Transition State Theory: New Enantio- and Diastereoselectivities

Nine efficient aldolase antibodies were generated by using hapten 1. This hapten unites reactive immunization and the transition state analogue approach in a single molecule. Characterization of two of these antibodies reveals that they are highly proficient (up to 1000-fold better than any other antibody catalyst) and enantioselective catalysts for aldol and retro-aldol reactions and exhibit enantio- and diastereoselectivities opposite to that of antibody 38C2.     

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa,and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

We present the MALDI‐TOF/TOF‐MS analyses of various hapten–bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer‐equipped, terminal monosaccharide of the O‐specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten–BSA neoglycoconjugates with different hapten–BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI‐TOF/TOF‐MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI‐TOF/TOF‐MS/MS of the glycated peptides. The product‐ion scans of the protonated molecules were carried out with a MALDI‐TOF/TOF‐MS/MS tandem mass spectrometer equipped with a high‐collision energy cell. The high‐energy collision‐induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y‐series product ions was very useful for the sequencing of various peptides. The series of a‐ and b‐product ions confirmed the sequence of the conjugated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of structural specificity in antibody—antigen reactions by capillary electrophoresis with laser-induced fluorescence detection

A rapid and simple procedure for screening antibodies for binding to an antigen is proposed. A fluorescent hapten —dye conjugate was prepared by labeling the amino moiety of the hapten with a commercially available reactive cyanine dye, Cy5 (excitation maximum: 650 nm, emission maximum: 670 nm). A fixed amount of the Cy5—hapten was titrated with serial dilution of the antibody. Each of the titration mixture was analyzed by capillary electrophoresis (25 cm × 20 μm column) monitored by laser-induced fluorescence (laser: 10 mW helium-neon, 632.8 nm). Free and antibody-bound Cy5-hapten were analyzed simultaneously on the electropherogram. Competitive immunoassay of hapten was demonstrated with low-end sensitivity of 5 · 10⁻⁸ M, about 10× more sensitive than the present drug screening methods. Using morphine as an example, the screening of various antibodies (from different vendors) and cross-reactivity of morphine analogues using the present procedure will be discussed.     

Direct hapten-linked competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of O-pinacolyl methylphosphonic acid

Immunoassay detection of O-pinacolyl methylphosphonic acid (PMPA) employing direct coating of N-2-aminoethyl-O-pinacolyl methylphosphonate (hapten B) on microtiter plates is reported. Coating was achieved by covalently linking hapten B to a glutaraldehyde (GA) polymer network directly bound to the polystyrene (PS) surface of a standard 96-well microtiter plate. 4-(2-(O-Pinacolylmethylphosphoryl amino)ethyl amino)-4-oxobutanoic acid (hapten A)-ovalbumin (OVA) conjugate served as the coating antigen for comparison with direct hapten B-coated plates in the CIELISA format. The developed assay employing direct hapten B coated plates demonstrated enhanced sensitivity with the IC(50) value for PMPA being 0.027 μg mL(-1). The assay could detect PMPA even at the concentration of 0.006 μg mL(-1). The mean recovery of standard PMPA (spiked in water) was found to be 83.7%.     

小分子抗原人工合成进展

小分子免疫分析技术的应用日渐广泛,合成稳定的、具有良好免疫原性的人工抗原是制备单克隆抗体和建立免疫分析方法的前提和关键。 本文对国内外半抗原的设计与合成方法、载体的选择、半抗原与载体的耦联方法等进行了综述,并对小分子抗原人工合成中相关的问题进行了讨论。     

Synthesis of a novel diarylphosphinic acid: a distorted ground state mimic and transition state analogue for amide hydrolysis

We describe herein the synthesis of a new unsymmetrical diarylphosphinic acid, a hapten aimed to produce catalytic antibodies for the hydrolysis of heterocyclic amides. The phosphinate functionality was selected as a mimic both of the tetrahedral intermediate and the transition state of higher energy along the reaction profile. The phenyl and 2,4,6-(trimethyl)-phenyl groups flanking the phosphinate were chosen in order to impose rotation around the P–C bond, a choice supported by ab initio calculations. This new hapten should elicit catalytic antibodies whose binding site could affect the distortion at nitrogen as well as the twist along the N–C(O) bond for heterocyclic amides. This hapten along with a series of new sterically hindered unsymmetrical phosphinic acid derivatives was prepared by a key palladium-catalysed step.     

吡虫啉的酶联免疫吸附分析方法研究

通过碳二亚胺法将吡虫啉交联于牛血清蛋白(BSA)作为免疫抗原,混合酸酐法将吡虫啉交联于卵清蛋白(OVA)作为包被抗原,免疫Balb/c小鼠,采用B细胞杂交瘤技术,经免疫、融合、筛选、克隆,得到抗吡虫啉单克隆抗体,抗体亚类为IgG1,制备的单克隆抗体效价达1×107,确定了吡虫啉酶联免疫吸附分析方法(ELISA)的最佳工作条件,建立了定量测定吡虫啉的间接竞争ELISA方法。本方法的IC50为(15.12±1.28)μg/L,检出限为(1.76±0.02)μg/L。与其它吡虫啉结构类似物无交叉反应。批内相对标准偏差为4.5%;批间相对标准偏差5.1%,饮用水、重庆理工大学地下水和重庆市花溪河地表水平均添加回收率分别为102%,97%和85%。本研究建立了一种快速检测环境水中吡虫啉残留的方法。     

Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines

A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman’s test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3–5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.     

特异性吗啡抗原和单克隆抗体的制备

吗啡分子的不同部位与蛋白偶联诱导出的抗体的专一性差异很大。为减少交叉反应,提高抗体分子对游离吗啡的特异性识别,选择吗啡分子N位进行修饰,合成了半抗原降吗啡,并设计和合成不同的连接臂,将半抗原用不同的连接臂与不同的载体蛋白共价结合分别制备了免疫抗原和筛选抗原,经细胞融合和筛选,成功地获得了5株可分汔抗吗啡单克隆抗体的细胞株：28H10,29D5,36G3,42D5,43C4。     

烟碱分子烙印聚合物的吸附特性

利用分子烙印技术，以烟碱为烙印分子，甲基丙烯酸(MAA)为功能单体，乙二醇二甲基丙烯酸酯(EDMA)为交联剂，合成了对烟碱具有特异性作用的分子烙印聚合物P(Nic)；通过平衡吸附实验，评价了其对烟碱的亲和力和选择性。与非烙印聚合物相比，P(Nic)对烟碱表现了很高的亲和力；Scatchard分析表明在P(Nic)中存在对烟碱有不同的亲和力的两类作用位点。通过与氨基吡啶类物质在P(Nic)上的吸附行为比较，表明P(Nic)对烟碱具有很好的选择性。本工作证明了用P(Nic)作为固相萃取(SPE)材料选择性地从烟草烟雾中提取烟碱的可能性。     

Preparation and Characterization of Olaquindox Polyclonal Antibody

本研究先将喹乙醇琥珀酸单酯化,使其转变为带有羧基的衍生物。合成产物经重结晶纯化,得率为52.47%。合成产物进行结构鉴定,合成了具有喹乙醇分子结构特征的喹乙醇半抗原。采用活化酯法将半抗原与牛血清白蛋白（BSA）进行偶联,采用混合酸酐法将半抗原与卵清蛋白（OVA）进行偶联,分别制备免疫原和包被抗原。紫外扫描与红外图谱分析结果表明,半抗原与载体蛋白偶联成功,与BSA、OVA的结合比分别为3.8:1和5.0:1。以OLA-BSA作为免疫原免疫4只新西兰大白兔,获得了较高效价的抗血清,以OLA-OVA作为包被抗原,间接ELISA法测定各兔的效价,分别为1:6400;1:1600;1:12800;1:6400。间接ELISA法喹乙醇的抑制中浓度IC50为743.3 ng/mL,最低检测限IC20为5.71 ng/mL。从电泳图谱分析可以看出,经纯化得到了纯度较高的喹乙醇多克隆抗体,为喹乙醇免疫分析方法的进一步研制和开发奠定基础。