Design and efficient synthesis of novel haptens and complete antigens for the AOZ,a toxic metabolite of furazolidone

A good strategy was brought forward for designing efficient haptens and complete antigens for 3-amino-2-oxazolidinone （AOZ）. Haptens designed newly were achieved facilely in good yield by using LiCI-N（Et）3 as new catalysis system, the structure of which was elucidated by spectroscopy analysis, such as NMR and MS. Novel antigens for AOZ were prepared successfully by convenient active ester method. The ratios of haptens 3 and 4 to carrier proteins were proven respectively as 41 ： 1 （5a）, 39：1 （6a）, 11：1 （5b） and 9：1 （6b） by trinitrobenzene sulfonic acid （TNBS） method. The results of indirect competitive ELISA （ic-ELISA） of antiserums indicated that the haptens with a short unsaturated side chain can evoke specific immune response effectively.

Characterization of Hapten-Protein Conjugates for Immunoassay of Polycyclic Aromatic Hydrocarbons(PAHs)

Preparation and characterization of the hapten-protein conjugates are fundamental to developing environmental immunoassays. As a hapten, 1-pyrenebutyric acid（PBA） was conjugated to the carrier protein of bovine serum albumin（BSA） or ovalbumin（OVA） by active ester method. Infrared spectra（IR） showed that PBA-BSA and PBA-OVA conjugates were successfully prepared. The number of the haptens conjugated to the carrier protein was determined by ultraviolet spectra（UV） and matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF-MS）. The calculated average binding ratios of PBA/BSA and PBA/OVA were 18：1 and 10：1 by UV, and 31：1 and 22：1 by MALDI-TOF-MS, respectively. Although there was a discrepancy between the results determined by the two methods, both of them were useful for the characterization of the hapten-protein conjugates. The antibody was produced against the antigen of PBA-BSA, and the affinity was tested by the double agar diffusion method The conjugates and the antibody could be used for developing a sensitive and selective immunoassay of polycyclic aromatic hydrocarbons（PAHs）.

A New Hapten for Immunoassay of Aldicarb

A new hapten, aldicarb oxime succinic ester （AOSE）, was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA （bovine serum albumin） conjugate. Enzyme-linked immunosorbent assays （ELISAs） showed that this antiserum had high affinity to aldicarb and can be used for sensitive and selective immunoassay of aldicarb.

Design and Synthesis of 6α-Corticosteroid Haptens and Their Bovine Serum Albumin (BSA) Conjugates

The site of attachment of protein carrier to corticosteroids has great influence on the specificity of produced antibody. In order to obtain highly specific and accurate antibodies for bioimmunoassay determination of cortisol, different tether lengths of 6-corticosteroid haptens and their BSA conjugates were designed and synthesized.

Studies on Increasing the Activity of Selenium-Containing Abzyme (Ⅰ)——Synthesis, Characterization and Properties of Hapten and Antigen

The thiol group of glutathione (GSH) reacts specifically with 2.4-di-m-trochlorobenzene to give S-substituted dinitrophenyl glutathione (GSH-S-DNP);two carboxyl groups of GSH-S-DNP were further esterified by n-butanol to produce the hapten,multisubstrate analog GSH-S-DNP Butyl Ester (GSH-S-DNP BE).The primary structure of the hapten was characterized by the free ammo group analysis,1H NMR,IR determinations and the elemental analysis.The hapten was then conjugated to bovine serum albumin (BSA) in the presence of glutaraldehyde.The reaction mixture was purified by Ultrogel AcA54 coluni chromatography to give the antigen.On an average,25 haptens were bound to each BSA molecule.Electrophoresis analysis showed that the average molecular weight of the antigen was 87 KD.CD spectrum showed that the α-helix content of the antigen increased.

Synthesis and Density Functional Theory(DFT) Calculation of Haptens for PAHs

Five PAHs haptens 3 a～3 e were synthesized from naphthalene, anthracene, chrysene, pyrene and acenaphthene via two steps including Friedel-Crafts acylation and WolffKishner-Huang reduction. The sixth hapten 4-(acenaphthylen-3-yl)butanoic acid(3 f) was prepared from hapten 3 e via three steps including esterification, bromination and elimination. Their structures were confirmed by melting point, ~1H NMR and ^(13)C NMR. Their optical properties and crystal structures were also investigated. The results of density functional theory(DFT) calculation provided the supports that the size, shape(geometry) and electronic properties at the corresponding parts of 3 a～3 f did not change significantly, compared to those of PAHs. The haptens 3 a～3 f were coupled with bovine serum albumin(BSA) to make antigens. The coupling ratios were 1:20～1:38. These results show that the haptens could be used to induce specific antibodies for PAHs.

Synthesis and Characterization of Specific Haptens Used to Generate Abzyme with Glutathione Peroxidase Activity

Glutathione was modified selectively by 2,4 dinitrochlorobenzene,giving S substituted dinitrophenyl glutathione(GSH S DNP).GSH S DNP was further esterified by iso butanol,hexanol,cyclohexanol and benzyl alcohol.Four haptens used to generate abzyme with glutathione peroxidase(GPX)activity were synthesized.They are GSH DNP biesters:GSH DNP di iso butyl ester(GSH DNP IBU),GSH DNP bihexyl ester(GSH DNP HE),GSH DNP bicyclohexyl ester(GSH DNP CH),GSH DNP bibenzylmethyl ester(GSH DNP BE).The structures of the haptens were characterized by means of elemental analysis,IR and 1H NMR.

Hydrophobic Pocket Modification and Humanized Catalytic Antibodies with Glutathione Peroxidase Activity(Ⅰ)——Preparation and Characterization of Haptenes and Conjugates

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Rational hapten design and establishment of broad-spectrum indirect competitive enzyme-linked immunosorbent assay for benzimidazoles monitoring in milk

Objectives Benzimidazoles(BZs)are commonly used for the treatment of soil-transmitted helminth infections in veterinary clinics;however,misuse and overdosing of BZs will cause residual problems and have the potential to damage human health through the food chain.Thus,the existence of BZs in foods needs more attention.This study aims to establish a broad-spectrum immunoassay for rapid detection and to simultaneously monitor BZs in milk.Materials and Methods Based on structure analysis,a‘zero epitope loss’strategy,which introduced a spacer arm into the imino group of the imidazole ring of albendazole,was first adopted for hapten modification to obtain an ultra-sensitive and broad-spectrum antibody.An indirect competitive enzyme-linked immunosorbent assay(icELISA)was established for the detection of 18 BZs in milk sample with a single-step pretreatment.A quantitative structure–activity relationship model was constructed to interpret and predict the recognition.Results The antibody could recognize 20 BZs and the half-inhibitory concentrations ranged from 0.054 to 417.58 ng/mL,the limits of detection of icELISA ranged from 0.4 to 89.4 ng/mL,and the mean recovery rates ranged from 76.49%to 120.40%,with a coefficient of variation<20%.Substituent R1 of BZs was considered to be the main influencing factor for recognition,and the comparative molecular field analysis model(q2=0.724,r2=0.998)was finally chosen for further prediction.Conclusions The results indicated that the established icELISA could simultaneously identify 18 BZs,with good accuracy and precision,which was suitable for rapid detection of BZs in milk.

Analysis on molecular interaction mechanism of four hapten flavonoids in Shuang-huang-lian powder injection with bovine serum albumin

Baicalein, baicalin, scutellarin and Chrysin-7-O-β-D-glucuronide are the major flavonoids of the Shuang-huang-lian powder injection. These flavonoids are thought to be haptens that can induce allergic reactions. The interaction mechanism of these haptens with bovine serum albumin （BSA） was investigated by surface plasmon resonance （SPR） and molecular modeling method. The SPR study indicated that these compounds could specifically bind to the BSA with one binding site and equilibrium dissociation constant （KD） values were determined. Molecular modeling explored the mechanism of interaction under simulated physiological condition. The result of molecular modeling indicated that flavonoids could bind with BSA in the hydrophobic pocket of sub-domain II with hydrogen bonding as the main acting force.

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 addition,we have also developed a simple method to prepare hapten 2 for the pyrethroid insecticide cyhalothrin,which only requires two steps including the reactions of nitration and reduction.Three haptens were coupled to carrier proteins to prepare the corresponding artificial antigens.The molecular structures of these three haptens were identified by 1H NMR and MS,and those of the artificial antigens were confirmed by UV-vis.

半抗原直接包被的四环素酶联免疫吸附分析法的建立

为建立更优的四环素(tetracycline,TC)酶联免疫吸附分析法(enzyme-linked immunosorbent assay,ELISA),将TC与蛋白质偶联制备人工完全抗原免疫BALB/c小鼠,获得抗TC的单克隆抗体(monoclonal antibody,MAb)。采用酸性高锰酸钾羧化酶标板,将TC直接包被在酶标板上,并对ELISA反应体系条件进行优化,构建一种半抗原直接包被的TC ELISA检测方法。所制备的MAb特异性良好,交叉反应率低。所构建的检测方法最低检测限IC_(10)值为0.306 ng/mL,半数抑制率IC_(50)值为9.26 ng/mL,牛奶和水中加标回收率分别为96.46%~101.89%、96.84%~102.50%。与人工完全抗原包被的检测方法相比(IC_(10)值:0.624 ng/mL,IC_(50)值:28.24 ng/mL,牛奶和水中加标回收率分别为93.86%~105.12%、94.04%~105.56%),检测灵敏度有明显提高,并可用于实际样品中TC含量的检测,具备良好的应用前景。

Synthesis and characteristics of a novel artificial hapten using the copper mercaptide of penicillenic acid from penicillin G for immunoassay of heavy metal ions

In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid （CMPA） derived from penicillin. Results from tests with immune rabbits indicate that： （i） A new antigen synthesized with CMPA has good stability and is safe for immunizing animals with no toxic phenomena being found in animal experiments; （ii） the immunogenic antigen （CMPA-BSA） can stimulate the immune system to produce specific antibodies with high titrations, up to 150000; and （iii） antibodies in antisera showed higher affinity to OVA-GSH-CuC1 than OVA-GSH, which indicates that the antibodies have specific affinity towards copper ions. These results confirm that the novel hapten and relevant antigen for copper ion have been successfully synthesized, giving progress towards an immunoassay for copper ions in environmental and food samples.

Preparation of hapten-specific monoclonal antibody for cadmium and its ELISA application to aqueous samples

High-affinity and specific monoclonal antibodies against cadmium-ethylene diamine tetraacetic acid(EDTA)complex have been produced using the hybridoma technique.A hapten was synthesized and characterized by Fourier Transform Infrared Spectroscopy(FT-IR)and UVVis.Competitive enzyme-linked immunosorbent assay(ELISA)for quantitative detection of cadmium in aqueous sample was developed.The monoclonal antibody with high level of binding affinity for Cd-IEDTA-BSA and high specificity for soluble Cd-EDTA complex showed less than 0.99%cross-reactivity with other 11 metals.The limit of detection was 0.10μg·L^(-1),and the effective linear range was 10^(-1)-10^(3)μg·L^(-1).The intra-and inter-assay coefficient variations were 1.5%-6.3%and 3.2%-7.4%,respectively.The spike recovery in different water samples were between 98.5%and 110.3%.The detection limit of this assay was well below the allowable concentration of cadmium(3μg·L^(-1)),and the working range was wider than that of other methods which showed the range of 2.19-86.38 and 0-10^(3)μg·L^(-1).The competitive ELISA established in this paper was sensitive and accurate in the screening of cadmium in aqueous samples.The results will lay a solid foundation for construction of an immunoassay kit for cadmium.

Staphylococcal enterotoxin B-derived haptens promote sensitization

T helper 2 （Th2） polarization is a major pathological feature in allergic diseases; its etiology is not fully understood. This study aims to elucidate the adjuvant effect of the microbial product-derived small peptides in the initiation of antigen-specific Th2 polarization. In this study, a clinical survey of patients with chronic rhinosinusitis （CRS） and food allergy （FA） was carried out. The Staphylococcal enterotoxin B （SEB）-derived small peptides （Ssps） were examined in the human stool extracts. The formation of Ssp/antigen adducts was tested in a protein-protein combination assay. The bone marrow-derived dendritic cells （BMDCs） were employed to test the role of Ssp/ovalbumin （OVA） adducts in the dendritic cell （DC） maturation. A mouse model was developed to test the role of Ssp/OVA adducts in the initiation of ＂l＇h2 polarization in the intestine. The results showed that 54 （18.2%} patients with FA were diagnosed among 296 patients with SEB＋ CRS; only eight （2.9%） FA patients were identified among 272 patients with SEB- CRS. Ssps were detected in the stool protein extracts from FA patients with SEB＋ CRS, but not in those with SEB- CRS. Ssp/OVA adducts induced DC maturation, speeded up DC migration, activated CD4＋ T cells in the regional lymph nodes and induced skewed Th2 polarization in the local tissue. We conclude that patients with SEB ＋ CRS are prone to suffering from FA. SEB can be degraded to Ssps in the gastrointestinal tract. The Ssps can bind macromolecular antigens to form adducts to promote the antigenicity of the antigens and induction of the antigen-specific Th2 polarization and inflammation in the local tissue.

牛奶中喹诺酮类药物残留的AlphaLISA检测方法的研究

本文旨在建立一种可检测牛奶中喹诺酮类药物(Quinolones,QNS)残留的均相光激化学发光免疫分析方法(Amplifiedluminescent proximity homogeneous assay linkedimmune sorbent assay,AlphaLISA)。采用EDC/NHS活泼酯法在诺氟沙星分子连接6-氨基己酸作为间隔臂,制备生物素修饰诺氟沙星半抗原(Biotin-6AS-NOR),并在受体微球上偶联QNS单克隆抗体,Biotin-6AS-NOR可与牛奶样品中的QNS竞争结合喹诺酮抗体;通过优化受体微球的QNS抗体偶联量,Biotin-6AS-NOR及供/受体微球的用量等参数,以及牛奶样品的稀释方案,建立了牛奶中QNS残留的AlphaLISA检测方法。采用1:5牛奶样品稀释方案,所建立牛奶中QNS残留的AlphaLISA检测方法的检出限和定量限分别为0.19 ng/mL和0.46 ng/mL,回收率在85.97%~110.11%之间,日内和日间变异系数均小于10%,与诺氟沙星、恩诺沙星、环丙沙星、氧氟沙星、培氟沙星、达氟沙星和洛美沙星的交叉反应率均高于70%。本研究所建立AlphaLISA检测方法具有灵敏度和准确度高、重复性好等优点,且在整个检测过程中不需要繁琐洗涤操作,具有较高的应用和推广价值。

Synthesis of haptens and production of antibodies to bisphenol A

Three immunizing haptens of bisphenol A(BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were located at different positions in BPA, and different length spacer arms were tested. Highly sensitive polyclonal antibodies were obtained and characterized using indirect competitive enzyme-linked immunosorbent assay(ic ELISA). Under optimized conditions, the half maximal inhibitory concentration(IC50) value of the best polyclonal antibody was 2.1 mg·L^(-1), based on coating heterogeneous antigens, and this optimal polyclonal antibody was highly sensitive toward BPA and displayed negligible crossreactivity with bisphenol B and bisphenol E. A sensitive ic ELISA method utilizing the polyclonal antibody was developed for the determination of BPA in milk. In spiked samples(5, 10 and 20 mg·L^(-1)), the recovery ranged from 80% to 102% with a coefficient of variation(CV) value below 15.8%. The limit of detection of ic ELISA was1.95 mg·L^(-1). These results indicate that the ic ELISA method is suitable for the detection of BPA in milk.

布鲁氏菌病M5、S2疫苗免疫血清半抗原-琼脂扩散试验方法检测评价

目的应用NH-AGID方法对布鲁氏菌M5、S2疫苗株免疫血清进行检测,评价该方法适用性。方法选取3~5月龄羔羊74只,随机分为3组。分别以S2株1.0×1010 CFU、5.0×109 CFU灌服和皮下注射接种羔羊各25只,M5株以1.0×10^(9) CFU皮下注射接种羔羊24只。免疫后7 d~150 d采集羊全血,分离血清,经RBT初筛和SAT确诊免疫抗体阳性血清。评估NH-AGID方法对M5和S2株疫苗免疫抗体与感染抗体的鉴别诊断特异性。结果NH-AGID方法对M5株疫苗免疫阳性血清的LPS抗体检出率为100%,NH抗体检出率8.3%~29.2%,鉴别诊断特异性为82.8%。对S2株疫苗口服组免疫阳性血清的LPS抗体检出率为31.2%~66.7%,NH抗体检出率为0%;皮下注射组免疫阳性血清的LPS抗体检出率为45.4%~100%,NH抗体检出率为4.5%~20%。对S2株疫苗免疫血清的鉴别诊断特异性为96.5%。结论NH-AGID方法不完全适用于羊用布鲁氏菌疫苗M5株和S2株的鉴别诊断,该方法的鉴别诊断特异性与疫苗毒力、免疫剂量和途径等因素相关,应进一步研究明确该方法的适用范围及条件,以期合理应用于布病防控诊断工作中。

有机氯类禁限用农药抗体的制备和应用研究进展

农药在防治病虫害、提高农产品产量和质量方面效果显著,但一些农药因高毒、致癌、致畸、致突变、代谢缓慢,对人类和环境产生不利影响而被我国禁止或限制使用。有机氯类禁限用农药作为其中产量最多、用量最大的一类,存在较高的健康或环境风险而备受关注。建立高效便捷的有机氯类禁限用农药残留的检测方法,对确保食品安全、助力绿色生态农业发展、打破食品进出口技术贸易壁垒具有重要意义。免疫分析法以其快速、简便、灵敏等优势被广泛应用于食品中农药残留的检测。本文综述了有机氯类禁限用农药免疫检测方法的研究进展,介绍了半抗原的合成和抗体的制备,总结了抗体在免疫检测中的应用,主要包括酶联免疫分析法、免疫层析试纸条法、化学发光法、荧光免疫分析法和免疫传感器,可对未来有机氯类禁限用农药免疫分析法的进一步研究开发提供参考。

三唑类杀菌剂的免疫检测技术研究进展

三唑类杀菌剂是一类有机含氮杂环类化合物,兼具保护、治疗作用而被广泛应用于水果、蔬菜、烟草等农产品生产中。近年来,随着食品物流行业发展,三唑类杀菌剂在采后保鲜、贮藏和运输领域的应用也越来越普遍。但其半衰期长、不易降解,容易造成食品中残留超标问题,影响人类健康和进出口贸易。因此,实现三唑类杀菌剂的高效快速检测,对食品安全和产业健康发展具有重要意义。利用抗原-抗体结合的免疫检测技术具有高效、灵敏等优点,被广泛应用于食品中农药残留的快速检测。该文以三唑类杀菌剂的免疫检测方法为出发点,总结了当前国内外三唑类杀菌剂的半抗原的设计合成、完全抗原和抗体的制备进展,并分类介绍了已开发的三唑类杀菌剂的免疫检测方法(主要包括酶联免疫分析法、免疫层析试纸条、荧光免疫分析法和免疫亲和色谱法),为今后开发更为快速简便的三唑类杀菌剂的残留检测方法提供参考。