伏马菌素B_1人工抗原的鉴定及抗体的制备

采用戊二醛(GA)一步法将半抗原伏马菌素B1(FB1)分别与鸡卵清蛋白(OVA)、牛血清白蛋白(BSA)偶联,制备2种具有免疫原性的FB1人工抗原FB1-OVA和FB1-BSA。分别采用凝胶电泳法、紫外光谱法(UV)、红外光谱法(IR)、基质辅助激光分析电离飞行时间质谱法(MALDI-TOF-MS)4种方法鉴定人工抗原偶联效果并测定偶联比。将FB1-BSA作为免疫抗原免疫BALB/c小鼠,FB1-OVA作为固相包被抗原,采用间接酶联免疫吸附法(i-ELISA)测定小鼠抗血清滴度。结果表明:4种方法均鉴定FB1人工抗原合成成功。凝胶电泳法测定FB1-OVA和FB1-BSA相对分子质量分别约为50 000和75 000。MALDI-TOF-MS测定FB1-OVA和FB1-BSA相对分子质量分别为48 009.212和74 355.301,并精确计算FB1-OVA和FB1-BSA偶联比分别为5∶1和10∶1。FB1-BSA免疫后小鼠的抗血清效价为1∶1.25×105,并具有较高灵敏度。     

伏马菌素B_1人工抗原的构建

碳化二亚胺法将伏马菌素B1(fumonisin B1,FB1)与载体蛋白进行偶联制备免疫抗原FB1-BSA及包被抗原FB1-OVA,比较了不同偶联条件对抗原合成的影响。紫外扫描初步显示载体蛋白与伏马菌素偶联成功。皮下注射免疫BALB/c小鼠,获抗FB1多克隆抗体,间接非竞争ELISA分析所得抗血清效价,最高可达1∶23000,间接竞争ELISA显示所制备的抗血清能有效识别伏马菌素B1,与脱氧雪腐镰刀菌烯醇(DON)、玉米赤霉烯酮(ZEN)、T-2毒素无交叉反应。     

伏马毒素B1单克隆抗体的制备及间接竞争ELISA方法的建立

采用戊二醛法将半抗原伏马毒素B1分别与载体蛋白钥孔血蓝蛋白（KLH）和蛋白卵清白蛋白（OVA）偶联成为完全抗原KLH-FB1和OVA-FB1。OVA-FB1作为包被抗原建立ELISA方法;KLH-FB1作为免疫原免疫8周龄Balb/c小鼠,应用杂交瘤细胞融合技术制备单克隆抗体。经三次亚克隆后,筛选出两株（6H3,6H11）能特异、稳定分泌抗FB1单克隆抗体的杂交瘤细胞株。选择其中一株杂交瘤细胞通过体内诱生腹水的方法制备单克隆抗体,经检测表明单克隆抗体的亚型为IgG1,轻链为κ型。采用间接ELISA测得腹水纯化后效价达l∶16 000,抗体对伏马毒素的半数阻断浓度（IC50）为8.26 ng/mL,对玉米赤霉烯酮、黄曲霉素B1等结构类似物几乎不存在交叉反应,与其他结构的氯丙嗪、链霉素等无交叉反应。利用获得的抗FB1单克隆抗体,建立了间接竞争ELISA检测方法,检测灵敏度为0.44 ng/mL,检测线性范围为0.93~73.06 ng/mL,加标回收率在线性范围内达到回收率可达78.4%~102%。     

伏马菌素B1胶体金免疫层析试纸条的研制

[目的]建立用于快速检测伏马菌素B1(FB1)的胶体金免疫层析试纸条。[方法]首先制备了FB1单克隆抗体和FB1-OVA偶联抗原,然后将FB1单克隆抗体与胶体金制成金标抗体包被在金标垫上,将FB1-OVA抗原和羊抗鼠Ig G包被在硝酸纤维素膜(NC膜)表面分别作为检测线和质控线。采用间接竞争法检测待检样品中的FB1与NC膜上的FB1-OVA抗原竞争结合金标抗体中的FB1单克隆抗体情况,并以检测线和质控线显示检测结果。[结果]本试验优化了金标抗体、检测抗原及羊抗鼠Ig G的包被量,检测FB1的灵敏度可达到2 ng·m L-1,最佳判定质量浓度为40 ng·m L-1。[结论]本试验制备的检测试纸条具有较高的特异性和稳定性,操作简单,检测时间仅需10 min,且与高效液相色谱法检测结果一致,可作为检测玉米中伏马菌素B1残留的有效手段。     

伏马菌素B1间接竞争ELISA检测方法的建立和应用

以人工合成的抗原OVA-FB1作为包被抗原,FB1(伏马菌素B1)为竞争抗原,两者与一定量的抗FB1单抗反应,建立检测FB1的间接竞争ELISA方法,对玉米样品进行初步检测应用。结果表明,间接竞争ELISA方法最佳抗原包被浓度为196 ng/ml,包被时间为4℃过夜,单抗最佳工作浓度为1∶4 000,酶标二抗最佳工作浓度为1∶2 500,最佳的抗原抗体竞争反应时间和温度为4℃过夜作用。可测最适范围为10~1 000 ng/ml,回归方程为y=1.113-0.313x,相关系数R2为-0.990,灵敏度为90.88 ng/nl,最低检测限为7.83 ng/ml,批内和批间变异系数分别为3.24%和2.45%,平均添加回收率为94.32%。     

1株抑制镰刀菌生长及产生伏马菌素链霉菌的分离和鉴定

【目的】从采自深圳近海红树林泥土中分离筛选到抑制再育镰刀菌生长和产生伏马菌素B1(FB1)的菌株,并对其进行鉴定,为控制食品中真菌毒素污染提供新途径。【方法】采用镰刀菌产毒素试验及生长对峙法,从采自红树林泥土中分离筛选目标菌株;根据菌株形态和培养特征、生理生化特性及16SrDNA序列分析,对目标菌株进行鉴定。【结果】从红树林泥土中分离到1株可抑制再育镰刀菌产生FB1和生长的菌株SZ1-6,初步确定该菌株属于卡伍尔链霉菌。菌株SZ1-6发酵液可显著抑制再育镰刀菌产生FB1,产毒抑制率达67%以上;菌株对再育镰刀菌生长也具有一定的抑制效果。【结论】筛选到1株对再育镰刀菌产FB1和生长均有抑制效果的链霉菌菌株SZ1-6,说明从海洋资源中寻找产抗生素的天然放线菌是可行的。     

伏马菌素B1诱导Marc-145细胞caspase非依赖型程序性细胞死亡

[目的]以Marc-145细胞为模型,研究了伏马菌素B1能否导致细胞产生其他类型程序性细胞死亡。[方法]利用caspase抑制剂、蛋白合成抑制剂、非凋亡程序性细胞死亡necroptosis抑制剂及ROS抑制剂,分别抑制caspase活性、蛋白质合成、necroptosis和ROS,采用结晶紫染色和AnnexinV-FITC/PI双染分析这些抑制剂对伏马菌素B1诱导Marc-145细胞死亡的影响。[结果]caspase、necroptosis抑制剂和2种自由基抑制剂不能抑制伏马菌素B1诱导的Marc-145细胞死亡,但蛋白合成抑制剂则可以显著抑制伏马菌素B1诱导的Marc-145细胞死亡。[结论]伏马菌素B1所诱导的Marc-145细胞的死亡是一种受基因表达调控的程序性细胞死亡,但与caspase、ROS和nec-roptosis无关。     

伏马菌素的研究进展

综述了伏马菌素毒性、测定方法及限量的研究现状。     

犬瘟热病毒单克隆抗体的制备及特性鉴定

用纯化的犬瘟热病毒免疫Balb/C小鼠,采用问接ELJSA方法筛选分泌阳性抗体的杂交瘤细胞株,挑选阳性孔进行亚克隆,用Vero细胞交叉反应试验及间接免疫荧光试验检测单抗的特异性.结果获得6株能稳定分泌抗犬瘟热病毒的单克隆抗体细胞株,分别命名为AD7、AE2、CE3、EH5、GG6和GF1.交叉反应试验及间接免疫荧光染色检测表明,6株单抗的特异性良好;经过体外连续传代及反复冻存、复苏,杂交瘤细胞均能稳定分泌特异性单抗.培养上清液及小鼠腹水ELISA效价分别为26～211和104～107.相加试验表明:单抗AE2、CE3、GG6和GF1具有共同的表位,而单抗AD7和EH5识别的位点与其他4株不同.     

伏马菌素的毒性及其作用机理

伏马菌素是主要由串珠镰刀菌和再育镰刀菌代谢产生的一类真菌毒素,其对农作物的污染在世界范围内普遍存在,尤其是玉米及玉米制品。在目前发现的多种伏马菌素中,伏马菌素B1是主要组分,且其毒性最大。本研究介绍了伏马菌素的化学结构、产毒菌株及在动物体内的代谢情况,着重概述了伏马菌素的毒性及其作用机理,并对今后如何减轻伏马菌素对人和动物的危害进行了探讨和展望。     

抗百菌清单克隆抗体的研制与鉴定

【目的】制备高灵敏、高特异性的百菌清(CTN)单克隆抗体,并对其免疫学特性进行鉴定。【方法】将CTN进行化学修饰后引入羟基活性基团,合成具有半抗原结构特征的百菌清衍生物（CTN-OH）;采用N, N-羰基二咪唑法（CDI）将CTN-OH与牛血清白蛋白（BSA）和鸡卵清蛋白（OVA）偶联合成人工免疫抗原CTN-BSA和包被抗原CTN-OVA,用紫外分光光度法（UV）和凝胶电泳（SDS-PAGE）进行鉴定后,免疫BALB/c小鼠,利用间接ELISA和间接竞争ELISA测定其多抗血清效价和敏感性,选出效价高、敏感性好的小鼠进行细胞融合,经过多次亚克隆筛选出分泌高敏感的特异性抗CTN单克隆抗体的杂交瘤细胞株,体内诱生腹水法制备抗CTN的单克隆抗体并对其免疫学特性进行鉴定。【结果】UV 和SDS-PAGE鉴定结果显示,CTN-BSA成功偶联,间接ELISA检测显示免疫的3只小鼠效价均达1﹕104以上,其中2号小鼠多抗血清敏感性最好,半数抑制浓度IC50为93.593 ng•mL-1,融合后筛选出4株杂交瘤细胞株1E8、1F4、2A10和2E2,其细胞上清效价分别为1﹕1.6×103 、1﹕6×102、1﹕6×102、1﹕6×102,1E8株腹水效价为1﹕5.12×105,抗CTN单克隆抗体对CTN的IC50为21.6685 ng•mL-1,且具有较好的特异性。【结论】本试验成功合成了CTN人工抗原,并制备了敏感性高、特异性好的单克隆抗体,为CTN免疫学快速检测方法的建立奠定了基础。     

Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.     

Preparation and Characteristic Identification of Monoclonal Antibody Against Sulfamethazine

Sulfamethazine is commonly incorporated into livestock feed as promotants of growth and for control of certain diseases in animals. It is retained in tissues of animals eating medicated diet. Consumption of meat from animals containing sulfamethazine may …     

利用Asia1型口蹄疫病毒VP1蛋白的单克隆抗体建立单抗竞争ELISA方法

1材料与方法1．1材料新生牛血清购自GIBICO生物公司;青霉素、链霉素购自哈尔滨制药六厂;降脂烷购自sigma公司;过碘酸钠购自上海生物试剂公司;脱脂乳购自飞鹤乳业公司;Babl／c小鼠购自兰州生物制品研究所。     

Development of Anti-Isoproturon Polyclonal Antibody

A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurca (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin(OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen,with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.0 × 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.07 mg mL-1.The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.     

利用Asia 1型口蹄疫病毒VP1蛋白的单克隆抗体建立单抗竞争ELISA方法(英文)

Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.     

非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位

【目的】制备非洲猪瘟病毒（African swine fever virus,ASFV）p30蛋白的单克隆抗体（monoclonal antibodies,MAbs）并初步分析其所识别的线性抗原表位,为ASFV及其抗体检测方法的建立及p30蛋白结构和功能的研究奠定基础。【方法】将原核表达并纯化的p30重组蛋白作为免疫原,免疫6—8周龄BALB/c雌鼠,每两周免疫1次,共免疫3次,首次免疫是抗原与等体积的弗氏完全佐剂乳化后免疫,第二次和第三次免疫与等体积的弗氏不完全佐剂乳化,3次免疫后1 w断尾采血,间接酶联免疫吸附试验（ELISA）检测血清抗体效价,选择血清效价最高的小鼠进行加强免疫,3 d后取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞按照4﹕1的比例使用PEG进行常规细胞融合。利用重组p30蛋白作为包被抗原,间接ELISA筛选阳性杂交瘤细胞,有限稀释法进行克隆纯化,直至筛出能够稳定分泌抗体的MAbs。将ASFV接种于猪肺泡巨噬细胞,以筛选的MAbs为一抗、兔抗鼠HRP-IgG为二抗,进行间接免疫荧光试验（IFA）。将感染和未感染ASFV的细胞沉淀处理后进行 SDS-PAGE并转印至硝酸纤维素膜,分别以IFA鉴定为阳性的MAbs上清为一抗、兔抗鼠HRP-IgG为二抗,进行Western blotting分析,筛选获得p30 MAbs。根据已知序列设计引物扩增p30ab与p30bc两段截短基因,其中p30ab代表由第86—153位氨基酸残基的截短体,p30bc代表由第120—187位氨基酸残基的截短体,原核表达部分重叠的截短p30蛋白,最终获得重组蛋白GST-p30ab与重组蛋白GST-p30bc。分别以GST-p30ab和GST-p30bc融合蛋白为包被抗原,以5株MAbs为一抗,以兔抗鼠HRP-IgG为二抗, 通过间接ELISA方法初步定位p30蛋白的抗原表位。【结果】以纯化的重组蛋白为包被抗原,经间接ELISA试验筛选出25株可分泌抗重组 p30蛋白的杂交瘤细胞株。IFA结果显示,5株MAbs（8F4、1D3、1H2、6C3和8E11）与ASFV感染的猪肺泡巨噬细胞IFA 试验呈阳性;Western blotting结果显示,5株MAbs均能够与ASFV感染的细胞呈阳性反应,与未感染病毒的细胞呈阴性反应。试验构建的p30截短体重组蛋白GST-p30ab以可溶和包涵体两种形式表达,而GST-p30bc仅以包涵体形式表达,以两组截短体融合蛋白为包被抗原,通过间接ELISA检测出MAbs 8F4、1H2和6C3与两个重组蛋白均能有效结合,证明MAbs 8F4、1H2和6C3抗原识别区域为两组截短蛋白重叠区域,即第120—153位氨基酸;MAbs 8E11与1D3则只能与GST-p30ab蛋白结合, 证明MAbs 8E11与1D3抗原识别区域为两个重组蛋白的非重叠区域,即第86—119位氨基酸。【结论】本研究可溶性地表达了p30蛋白的第86—153位氨基酸截短体重组蛋白,制备了5株p30 MAbs,定位到2个p30蛋白抗原表位。结合ELISA和IFA,可建立十分可靠的ASFV及其抗体的检测手段。     

多环芳烃-菲单克隆抗体制备及鉴定

为快速、准确检测环境中的菲(Phe),研究制备了抗菲单克隆抗体,并分析了抗菲单克隆抗体的免疫学特性。采用活泼酯法将2-菲丁酸与载体蛋白(BSA、OVA)共价连接制备免疫抗原(Phe-BSA)和检测抗原(Phe-OVA)。通过非变性聚丙烯酰胺凝胶电泳、紫外扫描图谱分析及计算偶联比验证偶联结果。采用足垫法免疫Balb/C小鼠,利用细胞融合技术、阳性杂交瘤筛选及克隆制备出抗菲单克隆抗体。结果表明:完全抗原制备成功,获得1株能稳定分泌抗Phe抗体的杂交瘤细胞株1G7,纯化后抗体效价为1∶6.4×105,亲和力常数为3.29×108L/mol。该单克隆抗体亚型为Ig G1,对Phe的IC50值为17.82 ng/m L,与5种多环芳烃具有交叉反应,可用于对环境中多种PAHs的广谱检测。