以免疫纳米微球为凝集原的犬细小病毒2型抗体凝集检测方法的建立

为建立以红色聚苯乙烯纳米微球为载体的犬细小病毒(CPV)抗体检测的间接凝集方法,本研究表达纯化了CPV-2VP2截短的重组蛋白rsVP2,并以rsVP2为致敏原与纳米微球偶联制备了免疫彩色纳米微球;通过对反应条件的优化,建立了一种快速检测CPV-2抗体的间接凝集方法。特异性试验结果显示,致敏微球仅与CPV-2阳性血清发生凝集反应,不与犬瘟热病毒、狂犬病病毒和犬腺病毒的阳性血清发生凝聚反应,特异性强;该凝集方法检测血清中和抗体的最低效价为1∶256;同一批次制备的3份致敏微球、3个不同批次制备的致敏微球以及4℃保存90d的致敏微球与CPV-2阳性血清的凝集反应特异性一致,凝集程度无明显差异,重复性稳定性均较好。该间接凝集检测方法对40份血清样品的检测结果与ImmunoComb■ Canine VacciCheck试剂盒(Dot-ELISA)检测结果的阳性符合率为97%。本研究建立的间接凝集方法为幼犬CPV-2母源抗体或免疫犬CPV-2抗体检测提供了一种快速、简便、准确、经济的方法。     

Serological cross-reactions between different brucella species and Yersinia enterocolitica. Agglutinating activity of 19 S and 7 S antibodies aginst somatic antigen of Brucella abortus and Yersinia enterocolitica type IX

Antisera from rabbits immunized with acetone-killed whole cells and with lipopolysaccharides from Brucella abortus (B.a.) and Yersinia enterocolitica type IX (Y.e.) were gel-filtered on Sephadex G-200 columns.All the antisera had serologically cross-reacting agglutinins against B.a. and Y.e. both in the 19 S and in the 7 S antibody fractions.The homologous and the heterologous agglutinating activity of 19 S and of 7 S antibodies was tested before and after treatment with dithiothreitol (DTT) and subsequent alkylation. Unlike 19 S antibodies, 7 S antibodies were resistant to the DTT reduction.The results from heterologous absorptions of the respective 19 S and 7 S antibody fractions indicated that 19 S antibodies both to B.a. and to Y.e. had a greater tendency towards being absorbed to the cross-reacting antigenic determinants than had the corresponding 7 S antibodies. The agglutination titres for 19 as well as 7 S antibody fractions derived from the immunizations with B.a. (both whole cells and LPS) fell more markedly after heterologous absorptions than did the analogous titres for corresponding Y.e. antibody fractions. Possible explanations of these differences are discussed.     

Seroprevalence for norovirus genogroups GII and GIV in captive non‐human primates

Noroviruses (NoVs) are a major cause of epidemic gastroenteritis in children and adults. Several pieces of evidence suggest that viruses genetically and antigenically closely related to human NoVs might infect animals, raising public health concerns about potential cross‐species transmission. The natural susceptibility of non‐human primates (NPHs) to human NoV infections has already been reported, but a limited amount of data is currently available. In order to start filling this gap, we screened a total of 86 serum samples of seven different species of NPHs housed at the Zoological Garden (Bioparco) of Rome (Italy), collected between 2001 and 2017, using an enzyme‐linked immunosorbent assay (ELISA) based on virus‐like particles (VLPs) of human GII.4 and GIV.1 NoVs. Antibodies specific for both genotypes were detected with an overall prevalence of 32.6%. In detail, IgG antibodies against GII.4 NoVs were found in 18 Japanese macaques (29.0%, 18/62), a mandrill (10.0%, 1/10), a white‐crowned mangabey (16.6%, 1/6) and in an orangutan (33.3%, 1/3). Twelve macaques (19.3%, 12/62), five mandrills (50.0%, 5/10), two chimpanzees (100%, 2/2) and a white‐crowned mangabey (16.6%, 1/6) showed antibodies for GIV.1 NoVs. The findings of this study confirm the natural susceptibility of captive NHPs to GII NoV infections. In addition, IgG antibodies against GIV.1 were detected, suggesting that NHPs are exposed to GIV NoVs or to antigenically related NoV strains.     

致仔猪过敏性大豆抗原蛋白β-conglycinin单克隆抗体的制备与鉴定

研究以人工合成的对应于β-conglycinin分子α'亚基上的一段抗原表位肽(RPQHPER,78-84α')作为半抗原,与载体蛋白(OVA)交联后,作为免疫原制备了致仔猪过敏性大豆抗原蛋白β-conglycinin的单克隆抗体(Mab 6G4),并应用酶联免疫吸附等方法进行鉴定。结果表明,制备的单抗Mab 6G4属于IgG1;该抗体能与大豆β-conglycinin特异性结合,IC50为4.7 ng/mL,并对β-conglycinin表现出较强的亲和力,亲和常数达6.9×109 L/mol。因此,制备的单克隆抗体Mab 6G4为分析大豆制品中β-conglycinin的含量及探讨β-conglycinin对仔猪的致敏机理提供了一个潜在的分析工具。     

抗大肠杆菌O157单克隆抗体制备过程中杂交瘤细胞的观测

采用细胞活力测定、形态学观察和遗传学检测的方法,研究了在用常规方法制备大肠杆菌O157单克隆抗体过程中,融合前后的细胞在细胞活力、细胞形态以及染色体数量的差异.结果表明,与凯骨髓瘤细胞相比,杂交瘤细胞的细胞活力和细胞形态无明显变化;当脾细胞与凯骨髓瘤细胞融合形成杂交瘤细胞后,其染色体数目接近两亲本细胞染色体数目之和.     

Production of monoclonal antibodies against AFLM (Ver-1), a middle key protein involved in aflatoxin biosynthesis

Aflatoxins are potent carcinogens, mutagens and teratogens, and are harmful to both humans and animals. As many as 30 genes are involved in aflatoxin biosynthesis. Among them, aflM (ver-1) gene was predicted to encode a 28-kDa NADPH-dependent ketoreductase (AFLM), which catalyzed middle enzymatic steps in aflatoxin biosynthetic pathway. AFLM (Ver-1) was proved to be necessary for conversion of versicolorin A (VERA) to demethylsterigmatocystin (DMST) in aflatoxin B₁ (AFB₁) biosynthesis. For these reasons, aflM gene was cloned and specific monoclonal antibodies for AFLM was developed to better define potential pathways of AFLM involved in AFB₁ biosynthesis. Monoclonal antibodies 11B2-1D7 and 3G5-4E7 were successfully screened out by immunizing mouse. Immunoblot analysis revealed that both had high sensitivity and specificity to identify native AFLM protein in A. flavus with detection limit of 11 ​ng/mL and 8 ​ng/mL respectively. These results showed that it was suitable for quantitative detection of AFLM in A. flavus isolate. Further investigation revealed that aflatoxin accumulations of various A. flavus were not dependent on AFLM biosynthesis. Overall, this is the first report for development for AFLM monoclonal antibody development and application in A. flavus quantitative detection.     

Confirmation of the passive transfer of maternal antibodies to calves following vaccination of pregnant cows with an inactivated Mannheimia haemolytica and Bovine herpes virus type 1 vaccine

The objective of this study was to evaluate the passive transfer of maternal antibodies to calves following vaccination of pregnant cows with an inactivated Mannheimia haemolytica (MH) and Bovine herpes virus type 1 (IBR) vaccine (Bovilis® MH + IBR). Sixty-two pregnant cows were allocated at random to two groups; one group was retained as a negative control group (T01), while the other group (T02) was vaccinated with Bovilis® MH + IBR on two occasions during their third trimester of pregnancy. Following calving, blood samples were collected from calves for the measurement of serum antibody titres to IBR and MH, with samples collected prior to suckling (Day 0) and on days 5 (±2), 14 (±3), 28, 56, 84, 112, 140, 168, 196, 224, 252 and 280. The group mean IBR blocking percentage remained low for T01 calves (calves born to T01 cows) between days 0 and 224 (range 4.5%–15.4%), while the group mean IBR blocking percentage increased for T02 calves (calves born to T02 cows) from 14.3% on Day 0 to 94.9% on Day 5 and remained significantly higher than T01 calves up until Day 252. The group mean MH titre (Log2) for T01 calves increased after suckling to 8.9 on Day 5, before declining and remaining stable (range 5.0–6.5). The group mean MH titre for T02 calves increased after suckling to 13.6 on Day 5 and then gradually declined; however, it remained significantly higher than T01 calves between days 5 and 140. Outcomes from this study have confirmed that colostral transfer of IBR and MH antibodies to newborn calves was successful and a high level of passive immunity was acquired by calves.     

猪流感病毒（A／swine／Jiangsu／2／2006（H3N2）NS1基因表达产物分析

采用RT—PCR方法克隆猪流感病毒H3N2亚型NS1全长基因,构建NS1基因原核表达质粒pET-28a—NS1和真核表达质粒p3XFLAG—CMV—NS1,在大肠杆菌和真核细胞内表达NS1基因,制备抗NS1多克隆抗体。用所制备的抗体分析p3xFLAG—CMV—NS1转染表达NS1蛋白和病毒感染细胞内的NS1蛋白。经终浓度为1mmol／L的IPTG诱导后,重组蛋白NS1在大肠杆菌中得到表达。表达蛋白纯化后免疫Wistar大鼠制备抗NS1蛋白多克隆抗体,Western—blot分析表明抗NS1多克隆抗体可以识别大肠杆菌表达的NS1蛋白、转染Veio细胞表达的NS1蛋白和病毒感染细胞内的NS1蛋白。间接免疫荧光发现NS1蛋白主要定位于细胞核。结果显示,本试验成功构建了NS1基因原核和真核表达系统,获得了NS1特异性多克隆抗体,分析了NS1细胞定位,为进一步研究NS1蛋白在病毒复制过程中的生物学功能和猪流感病毒的复制机理奠定基础。     

WCA-ELISA检测猪链球菌2型抗体

本试验应用菌体包被间接ELISA分别对70份假定阳性血清和70份假定阴性血清进行1:100、1:200、1:400、1:800稀释检测,利用血清流行病学原理对检测结果进行分析,确定了检测方法的最佳单一稀释度,以及判定阳性血清和阴性血清的OD值。并利用建立的间接ELISA对1997年采集的江苏地区的猪血清进行了检测。试验结果发现在1998年猪链球菌大暴发的前期,阳性率高达48%,尤其是双甸马塘地区,阳性率高达72.4%。