赭曲霉毒素A人工抗原的合成与鉴定

采用N-羟琥珀酰亚胺酯（N-hydroxysuccinimide NHS）法将赭曲霉毒素A（OTA）与牛血清白蛋白（BSA）偶联制备了免疫抗原OTA-BSA,采用1-乙基-3（3-二甲基氨基丙基）碳二亚胺（EDC）法将赭曲霉毒素A（OTA）与卵清白蛋白（OVA）偶联制备了包被抗原OTA-OVA。紫外扫描和SDS-PAGE凝胶电泳结果初步表明偶联成功。用OTA-BSA分10μg/只和50μg/只两个剂量分别免疫BALB/C小鼠,获得了高效价的特异性多克隆抗血清,效价可达1∶104,抑制效价达14.7ng,证明偶联成功,并显示低剂量免疫可以提高小鼠pAb的敏感性。本研究为OTA单抗的制备及其免疫学分析方法的建立奠定了基础。      

赭曲霉毒素A的研究进展

赭曲霉毒素A主要由赭曲霉和青霉属的某些菌株产生,广泛分布于谷物、其他植物性食品及相关产品和动物性食品,对动物和人体具有肾脏毒性、肝脏毒性,另外还有致畸、致突变和致癌作用,并有免疫抑制作用.随着对其研究的不断深入,现在愈来愈受到人们的关注,本文就赭曲霉毒素A对食品的污染、毒性、检测方法等方面进行综述.     

赭曲霉毒素A检测方法的研究进展

赭曲霉毒素A(ochratoxin A, OTA)是一种具有极端毒性、污染广泛及危害严重的次级代谢产物。由于赭曲霉毒素A具有这些特点且食品安全问题越来越被人们广泛关注, 所以确定食品和商品中的OTA污染水平非常重要。目前对于OTA的检测, 已经建立了非常多的分析手段, 如薄层色谱法(thin layer chromatography, TLC)、高效液相色谱法(high performance liquid chromatography, HPLC)、酶联免疫技术(enzyme linked immunosorlent assay, ELISA)、时间分辨荧光免疫分析技术(time-resolved fluoroimmunoassay, TRFIA)等。本文综述了多种赭曲霉毒素A的检测分析方法, 简介了它们的优缺点及研究进展, 并进行了比较, 以期为食品中赭曲霉毒素A的检测研究提供一些参考。     

ELISA法检测赭曲霉毒素A的改进研究

针对ELISA法检测赭曲霉毒素A(OTA),使用微振荡酶标板可以缩短免疫反应检测时间约30min。采用ELISA方法检测小麦样品中的OTA时,使用空白样品提取液来配制OTA标准溶液可以消除样品基质造成的干扰。同时对提取溶剂加以改进,采用NaCl-水-甲醇(10%:70:30)作为提取溶剂的提取效率最高。0.1、1、10μg/kg三个浓度水平的加标回收率为95%～110%,方法的重现性很好,变异系数小于10%。     

赭曲霉毒素A直接竞争ELISA试剂盒的研制

在多克隆抗体的基础上研制了赭曲霉毒素A(OTA)直接竞争酶联免疫检测(cd-ELISA)试剂盒.在0 ～ 10 ng/mL范围内,该试剂盒50％抑制率(IC50)为1.09 ng/mL,检测灵敏度(IC15)为0.08 ng/mL;与赭曲霉毒素B、C的交叉反应率分别为6.28％和0.16％,而与黄曲霉毒素B1等生物毒素未见有交叉反应;板内与板间平均变异系数分别为2.21％和2.79％;花生、玉米和玉米粉3种样品中OTA的检测低限分别为1.71,1.26和1.85 μg/kg,平均添加回收率在83.80％～ 91.40％;与HPLC检测方法具有较高的相关性,相关系数(R2)分别为0.94、0.88和0.90;检测时间只需20 min,可用于花生、玉米及玉米粉中OTA的批量快速筛查.     

免疫学法检测赭曲霉毒素A研究进展

赭曲霉毒素A是曲霉属和青霉属某些真菌次级代谢产物,其毒性大、污染范围广、与人类健康关系密切。对赭曲霉毒素A检测是利用其本身荧光特性进行高灵敏度荧光检测,易受其它本底物荧光干扰,造成检测周期长,成本高;免疫化学分析具有高度特异性、灵敏性和快速简便等优点,在赭曲霉毒素A检测中应用广泛,其中,免疫传感器具有独特专一性和选择性,在赭曲霉毒素A检测领域能发挥更大优势。     

食品中赭曲霉毒素A检测方法研究进展

赭曲霉毒素A(ochratoxin A, OTA)是曲霉属和青霉属等有毒真菌产生的一类次级代谢产物,是常见污染食品的五大真菌毒素之一,具有较强的肾毒性、肝毒性、神经毒性和免疫毒性,以及致畸、致癌和致突变作用。OTA广泛存在于各种谷物及其制品、葡萄与葡萄酒、咖啡等多种食品原料及其成品中,严重威胁人体健康,因此有必要建立快速、准确、灵敏的OTA检测方法。针对食品中OTA的检测,目前已经拥有许多方法,如薄层色谱法,高效液相色谱法,液相-质谱联用法以及酶联免疫吸附法等。本研究对赭曲霉毒素A不同检测方法的原理、优缺点等进行归纳总结,旨在为食品中OTA的检测提供支持。     

葡萄中碳黑曲霉的分离及其产生赭曲霉毒素A的研究

碳黑曲霉(Aspergillus carbonarius)是葡萄中产生赭曲霉毒素A(Ochratoxin A,OTA)的重要菌株.采集烟台赤霞珠(Cabernet sauvignon)葡萄,接种于孟加拉红培养基,从中分离到7株黑曲霉群(Aspergillus section black group)真菌,其中3株鉴定属于碳黑曲霉种,占黑色曲霉的43.8%.此3菌株分别接种在粮粒培养基上,静置培养,全部产生OTA,最高浓度达到1300μg/kg,而且,其中2株碳黑曲霉菌株在可可浆培养基上产生荧光,而从葡萄样品中未检出OTA.     

稻米中黄曲霉毒素和赭曲霉毒素A的研究进展

真菌毒素是由某些丝状真菌产生的有毒代谢产物,在自然界中普遍存在,严重威胁人畜健康。综述了黄曲霉毒素和赭曲霉毒素A的理化性质及危害,在稻米中的污染现状、限量标准及分析方法等方面进行阐述,希望能给真菌毒素的研究提供一些参考。      

抗赭曲霉毒素A单克隆抗体的制备及免疫学鉴定

通过碳化二亚胺法(EDC法)制备免疫原OTA-BSA,并免疫小鼠,经杂交瘤技术建立产抗赭曲霉毒素A(OTA)单克隆抗体的杂交瘤细胞株1株。通过体内诱生腹水法制备腹水,纯化后对其亚型、效价、灵敏性、亲和性、特异性等免疫学特性进行鉴定;单抗亚类为IgG1型,间接ELISA效价1:6.4×105,IC50为112.8pg/mL,亲和常数为9.74×1011L/mol,与赭曲霉毒素B交叉反应率为5.6%,与其他常见真菌产物交叉小于0.003%;本实验制备的抗OTA单克隆抗体具有高效、敏感、特异等特点,适合OTA的免疫学快速检测。     

盐酸可乐定人工抗原的合成及鼠源多抗血清ELISA鉴定

目的：获得敏感性高,特异性强的新型瘦肉精盐酸可乐定（Clonidine hydrochloride,CLO）鼠源多抗血清。方法：以CLO的衍生物盐酸阿可乐定（Apraclonidine hydrochloride,ACLO）为半抗原,采用戊二醛法将ACLO分别与牛血清白蛋白（Bovine serum albumin,BSA）和鸡卵清蛋白（Ovalbumin,OVA）偶联,合成免疫抗原CLO-BSA和包被抗原CLO-OVA。采用紫外扫描和聚丙烯酰胺凝胶电泳鉴定其偶联效果后,免疫BALB/c小鼠,制备多抗血清。利用间接ELISA和间接竞争ELISA鉴定其免疫学特性。结果：合成的人工抗原免疫效果较好,所获小鼠多抗血清效价均达到1∶12 800以上,其中3号小鼠敏感性最好,半数抑制质量浓度（IC₅₀）为82.75 ng/mL,与其他几种常见的瘦肉精类药物的交叉反应率均小于0.9%,特异性良好。结论：通过戊二醛法成功合成了高免疫原性的CLO人工抗原,并获得敏感性高,特异性强的CLO鼠源多抗血清。     

动物源食品中残留己烯雌酚免疫原的制备与表征

己烯雌酚（diethylstilbestrol,DES）为人工合成的二苯乙烯类雌性激素药。本文采用碳二亚胺法将其与牛血清白蛋白交联,经紫外扫描估算其交联率为22,SDS-PAGE实验初步表明,DES与BSA有交联。双向免疫琼脂扩散实验检测经DES-HS-BSA多次免疫的兔子的血清,结果表明,兔子产生了针对小分子DES的抗体,进一步说明了DES免疫原合成成功。      

甲胺磷人工抗原的合成与鉴定

采用重氮法直接用甲胺磷连接牛血清白蛋白合成了人工抗原。通过鉴定,该人工抗原的紫外吸收光谱呈现出与半抗原、载体蛋白的紫外吸收光谱均不同的吸收峰;红外光谱图中也同时呈现了半抗原与载体蛋白的特征吸收带;并测得人工抗原中甲胺磷与载体蛋白的结合比为15∶1,表明该人工抗原合成成功。     

土霉素完全抗原的制备与鉴定

为了建立土霉素完全抗原的制备方法,实验以土霉素为半抗原,牛血清白蛋白(BSA)为载体蛋白,利用N,N,-二环己基碳化二亚胺(DCC)、邻联甲苯胺(o-tolidine)为偶联剂,将牛血清白蛋白与土霉素偶联制备土霉素人工完全抗原。通过偶联率的计算确定邻联甲苯胺为最佳偶联剂。随后利用红外光谱分析和免疫原性测定实验鉴定了土霉素、牛血清白蛋白和邻联甲苯胺的偶联情况。结果表明,制得的土霉素完全抗原在免疫健康的Balb/c小鼠体内能刺激小鼠机体产生最高效价为25600(ELISA)的抗血清。     

Reduction of ochratoxin A in dry-cured meat products using gamma-irradiation

This study investigated the efficiency of gamma (γ)-irradiation in the reduction of ochratoxin A (OTA) present in dry-cured meat products prepared from intentionally contaminated raw materials from OTA-treated pigs. OTA concentrations determined in the samples (n = 24) ranged from 25.8 μg kg^–1 in bacon to 17.8 μg kg^–1 in smoked ham. After γ-irradiation at doses of 3, 7 and 10 kGy (i.e. the doses used in the food industry), a dose-depended OTA reduction was observed; however, it was not statistically significant. The mean OTA reduction achieved with 3-, 7- and 10-kGy γ-doses was approximated to 8.5%, 13.9% and 22.5%, respectively. The storage of irradiated samples (1 month, 4°C) did not significantly affect OTA levels. Based on the correlation between the OTA reduction level and basic chemical composition of dry-cured meat samples, OTA reduction may be linked to the samples’ fat content. The results indicate that γ-irradiation can reduce OTA levels in dry-cured meat products, but only to a limited extent due to the complexity of the matrix.     

Synthesis and identification of artificial antigens for cadmium and copper

Cadmium and copper were conjugated to two carrier proteins using bifunctional chelators, including derivatives of ethylenediamine N,N,N′,N′-tetraacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, to make artificial antigens for cadmium and copper. The artificial antigens were identified by nondenaturing gel electrophoresis, ultraviolet spectrophotometry, and graphite furnace atomic absorption spectrometry. Nondenaturing gel electrophoresis results revealed that the conjugate band migrations were different from those of the chelator-protein conjugates and carrier proteins alone. The ultraviolet spectrophotometry results revealed that the maximum absorption peak of the conjugates had only a little peak shift. The graphite furnace atomic absorption spectrometry results revealed that the metal content of the conjugates was much higher than that of the carrier proteins and chelator-protein conjugates. The results indicated that the artificial antigens for cadmium and copper were successfully synthesised and could be useful as immunising antigens.     

2,4-二氯苯氧乙酸人工抗原合成及鼠源多克隆抗血清的制备

采用碳化二亚胺法(EDC法)将2,4-D与牛血清白蛋白(BSA)及卵清蛋白(OVA)偶联,合成免疫原2,4-D-BSA和包被原2,4-D-OVA,采用紫外扫描(UV)和聚丙烯酰胺凝胶电泳(SDS-PAGE)法进行鉴定;用2,4-D人工免疫抗原(2,4-D-BSA)免疫BALB/c纯系小鼠,采用间接ELISA测定多抗血清效价、阻断ELISA鉴定其抑制。结果表明:BSA与2,4-D偶联后,波峰出现右移,表明偶联成功;SDS-PAGE电泳中BSA的泳动速度大于2,4-D-BSA,表明2,4-D已与BSA成功偶联;6只小鼠血清效价均达到了1:1.28×104,且1号小鼠多抗血清敏感性最好,半数抑制IC50为72.48ng/mL,表明成功获得了高效价、特异性好、亲和力较高的鼠源抗2,4-D多克隆抗体血清,为2,4-D残留免疫学检测方法的建立奠定了良好的基础。     

Co-occurrence of ochratoxin A and citrinin in unprocessed cereals established during a three-year investigation period

The aim of this study was to investigate ochratoxin A (OTA) and citrinin (CIT) co-occurrence in different unprocessed cereals (n = 189) originating from Croatia during a three-year investigation period (2014–2016) using validated enzyme immunoassay (ELISA) methods. CIT and OTA were determined in 49% and 7% of samples, respectively. Significantly higher (p < 0.05) overall mean concentrations were determined for CIT (66.8 ± 76.0 µg/kg) in comparison to OTA (5.2 ± 1.1 µg/kg). Based on the analysis of all investigated cereals, CIT was found about 15 times more frequently than OTA and in similarly (15-fold) higher concentrations, irrespective of the cultivation year. The results revealed a moderately positive correlation between OTA and CIT concentrations in maize (r_s = 0.44) and wheat (r_s = 0.59), whereas in barley and oat this correlation (p > 0.01) was not significant.     

伏马菌素B2人工抗原的制备研究

为制备伏马菌素B2人工抗原,通过戊二醛法和碳二亚胺法将伏马菌素B2偶联到栽体蛋白上,人工抗原免疫BALB/c小鼠后,酶联免疫吸附法测定抗血清的效价,竞争酶联免疫吸附法测定人工抗原的免疫原性.通过氨基化和羧基化的苏丹红作为内参,紫外可见光谱扫描图谱鉴定伏马菌素B2成功偶联到载体蛋白上,免疫原性鉴定证明,碳二亚胺法制备的人工抗原可刺激BALB/c小鼠产生抗伏马菌素B2的抗体.     

An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies

An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml^–1. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml^–1, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml^–1. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.