金黄色葡萄球菌肠毒素单克隆抗体制备与应用

目的制备抗B型金黄色葡萄球菌肠毒素单克隆抗体。方法纯化B型金黄色葡萄球菌肠毒素(SEB)免疫BALB／C小鼠的脾细胞与SO2／0骨髓瘤细胞融合，经筛选及克隆化，共获得6株能稳定分泌抗B型金黄色葡萄球菌肠毒素单克隆抗体的杂交瘤细胞株并进行了鉴定。结果6株单克隆抗体除两株属于Ig G2A外，其余均属于IGG1亚类，其培养上清和腹水的滴度分别为1：256～1：512和1：12800～1：25600，6株单克隆抗体均不与SEA发生交叉反应，仅有2株与SEC1有弱交叉反应。WESTERN-BLOT实验显示出一条特异性带，位于分子量28.5KDA处．证明了其特异性。结论成功获得抗B型金黄色葡萄球菌肠毒素单克隆抗体，为检测SEB打下了基础。     

大鼠抗肠道病毒71型衣壳蛋白VP1特异性中和单克隆抗体的研制与应用

目的研制大鼠抗肠道病毒71型（EV71）衣壳蛋白VPl特异性中和单克隆抗体（单抗）,并用Westem印迹、ELISA和免疫荧光检测（IFA）等方法验证其特异性。方法采用单克隆杂交瘤技术人工合成包含EV71 VP1中和抗原决定簇位点SPT0的多肽偶联载体蛋白KLH,腹腔内注射免疫大鼠数次后,取有预期免疫应答的脾细胞与SP2／0小鼠骨髓瘤细胞融合,用间接ELISA法筛选和有限稀释法亚克隆获得稳定的阳性单克隆杂交瘤细胞株并鉴定其IgG亚型。选取其中1株单克隆杂交瘤细胞系BC6,体外培养后注射于SCID裸鼠腹腔制备腹水以产生大量抗体,用ProteinA层析柱亲合纯化后获得纯化抗体。细胞培养微量中和试验测定该单抗中和EV71的滴度,并将其用于多种方法检测EV71特异性抗原。结果共获得8株稳定的单克隆杂交瘤细胞系,分别有6株属于IgGl亚型,2株为IgG2a亚型。通过制备腹水大量产生抗EV7l大鼠单抗BC6并纯化,其对EV71的中和滴度为1：32。BC6用于Western免疫印迹可特异检测大肠埃希菌表达的EV71VPl重组蛋白和EV71样品中的VPl,用于ELISA可灵敏特异地定性或定量检测EV71抗原含量,用于IFA可特异识别被感染Vero细胞内的EV71颗粒,而对柯萨奇病毒A16型无交叉反应。结论成功制备获得了大鼠抗EV71VPl的特异性中和单抗,可应用于Western免疫印迹、ELISA、IFA等方法,成为灵敏特异地检测EV71VPl或全病毒颗粒的有效工具。     

B型肉毒毒素单克隆抗体杂交瘤株的建立

[目的]制备、筛选稳定分泌B型肉毒毒素单抗的杂交瘤克隆,为相关研究提供工具。[方法]采用纯化的B型肉毒毒素免疫BALB/c小鼠,用免疫小鼠的脾细胞与骨髓瘤细胞Sp2/0融合,筛选阳性克隆。[结果]得到10株稳定分泌B型肉毒毒素单抗的杂交瘤克隆,1株为IgM,其余9株分泌IgG,亚类为IgG2a。10株单克隆抗体均与B型肉毒毒素呈特异性反应,而与标准A型肉毒毒素无交叉反应,腹水单抗的效价均在105以上。[结论]10株杂交瘤细胞株均能分泌抗B型肉毒毒素单克隆抗体,为B型肉毒毒素的检测和研究提供了工具。     

汉坦病毒G2囊膜蛋白基因的克隆及其免疫原性的初步研究

目的 构建汉坦病毒G2囊膜蛋白基因真核表达载体，进一步研究其免疫效果。方法 采用PCR方法扩增出G2基因，亚克隆于pcDNA3．1／HisB载体，重组阳性克隆进行酶切和测序鉴定。将重组质粒免疫BALB／C小鼠，用间接免疫荧光法(1FA)检测免疫小鼠血清中抗汉坦病毒76～118株的交叉抗体。结果 5只免疫小鼠血清中均检测到特异性的抗汉坦病毒76～118株的交叉抗体，与对照组相比有显著性差异，其滴度为1：10．99。结论 G2囊膜蛋白基因构建成功并能刺激机体产生特异性抗体，但其效价不高，该研究结果为研制有效的HFRS核酸疫苗奠定一定的实验基础。     

具备中和中东呼吸综合征冠状病毒活性的单克隆抗体的制备

目的制备并鉴定对中东呼吸综合征冠状病毒(MERS-CoV)具有中和活性的单克隆抗体,为进一步开展MERS-CoV感染与免疫保护机制研究,以及发展诊断与治疗手段奠定基础。方法 MERS-CoV S蛋白受体结合区与人IgG Fc片段融合表达的重组蛋白免疫BALB/c小鼠,将免疫后小鼠脾细胞与SP2/0骨髓瘤细胞融合,通过ELISA筛选出阳性克隆,随后通过多次亚克隆筛选出稳定分泌特异性抗体的杂交瘤细胞株,制备单抗腹水并进行抗体效价测定及亚类鉴定;通过MERS-CoV假病毒及活病毒中和试验筛选出中和单抗。结果获得了10株能够稳定分泌抗原特异性单抗的杂交瘤细胞株。制备的腹水单抗亚类均为IgG1;其中7株单抗抗体ELISA效价达到10 000以上,并有1株单抗对MERS-CoV具有良好的中和活性。结论本研究获得了1株对MERSCoV具有良好中和活性的单克隆抗体。     

Ⅱ型登革病毒非结构蛋白NS1的克隆表达及抗体制备

目的克隆表达Ⅱ型登革病毒（dengue virus 2，DV2）非结构蛋白1（DV2-NS1）基因，并制备其单克隆抗体。方法提取Ⅱ型登革病毒的RNA，扩增其NS1全长基因片段。经TA克隆将NS1基因克隆至pQE30载体中进行诱导表达，表达产物经变性处理后用Ni柱亲和层析纯化并复性，经免疫印迹法（Western Blot）鉴定抗原性。用复性后的NS1蛋白和灭活Ⅱ型登革病毒交替免疫Balb／c小鼠。取小鼠脾细胞与小鼠骨髓瘤细胞融合，杂交瘤细胞经间接ELISA法、间接免疫荧光法（IFA）进行单抗亚类、特异性的筛选与鉴定。结果携带重组质粒pQE30／DV2-NS1的菌株经诱导，可高效表达重组DV2-NS1蛋白，经复性获得可溶性蛋白，Western Blot证实重组的DV2-NS1蛋白具有抗原性；用重组蛋白和病毒混合免疫，共获得10株抗NS1蛋白的单抗，其中3株为特异性抗DV2-NS1蛋白，与其他3型登革病毒无交叉，另7株为混合交叉型；亚类分析一株为IgG2b，余为IgG1。结论获得重组Ⅱ型登革病毒非结构蛋白NS1，并得到10株抗DV2-NS1蛋白的杂交瘤细胞株，为进一步研究NS1的生物学特性和临床诊断试剂建立奠定了基础。     

登革热监测和单克隆抗体细胞株的建立和应用

1986年我区首次从病人分离到9株登革热Ⅱ型病毒，采用杂交瘤细胞融合技术获得6株抗Ⅱ型登革病毒单克隆抗体杂交瘤细胞株，其中4株产生型特异性高滴度单克隆抗体，并建立单抗IFA快速诊断方法，研制出单抗IFA诊断试剂盒。解决了不同型间的交叉反应，可用于型别鉴定，方法简便、敏感特异，与常规血清学试验鉴定毒株型别相比，可大大缩短时间而达到快速诊断和鉴定毒株的目的。     

西尼罗病毒单克隆抗体的制备与鉴定

目的制备西尼罗病毒单克隆抗体并对其生物特性进行鉴定,为快检试剂盒的研制奠定基础。方法通过动物免疫、细胞融合、克隆和筛选等方法制备西尼罗病毒单抗,应用间接免疫荧光(IFA)、ELISA方法进行特异性、效价、亚型等的鉴定。结果获得了5株西尼罗病毒的单克隆抗体,其中4株与所检的登革病毒1-4型、黄热病毒、乙脑病毒、布尼安病毒、基孔肯雅病毒均无交叉反应,1株与乙脑病毒有交叉反应;亚型分类均为IgG2a;效价可达1∶12 800以上。结论本研究获得了5个高滴度、特异性较好的西尼罗病毒单克隆抗体。     

环丙沙星单克隆抗体制备、纯化及鉴定

目的制备抗环丙沙星单克隆抗体.方法用碳二亚胺(EDC)法合成的环丙沙星-牛血清白蛋白完全抗原(CPFX-BSA)免疫BALB/c小鼠,通过杂交瘤技术筛选分泌抗环丙沙星单克隆抗体的杂交瘤细胞株.结果筛选出3株分泌抗环丙沙星单克隆抗体的杂交瘤细胞株1D10、3C6和3G7,其抗体类型分别为IgM、IgG1和IgG1.其中3C6腹水抗体纯度、抗体浓度、亲和常数分别为80.04%、3.96 g/L和1.01×108 L/mol.该单抗与恩诺沙星、诺氟沙星的交叉反应率分别为125.89%、19.50%,与青霉素、卡那霉素和庆大霉素等无交叉反应.结论该单抗可用于动物性食品中CPFX残留ELISA检测方法的建立.     

新疆出血热北疆株单克隆抗体杂交瘤细胞株的建立

作者应用新疆出血热病毒北疆株９０－９株鼠脑免疫ＢＡＬＢ／Ｃ小鼠，取脾细胞与ＳＰ２／０骨髓瘤细胞融合，共获得４株分泌抗ＸＨＦＶ北疆株单克隆抗体（ＭｃＡｂ）杂交瘤细胞系。核型分析表明符合杂交瘤细胞的标准。用间接免疫荧光法及间接酶联免疫吸附试验检测表明，ＭｃＡｂ的腹水具有很高滴度，其中２株可达５×１０５（ＥＬＩＳＡＰ／Ｎ≥２．１）。ＭｃＡｂ免疫球蛋白亚型鉴定均属ＩｇＧ，其中３株为ＩｓＧ１，１株为ＩｇＧ２ａ。对ＭｃＡｂ的初步应用表明，在反向被动血凝抑制试验（ＲＰＨＩ）中，分别采用南、北疆ＭｃＡｂ致敏血球检测小鼠腹水，其结果有明显差异。     

Development of inactivated vaccine against virus causing haemorrhagic fever with renal syndrome

B-1 virus belonging to the hantavirus group was serially passaged in the brains of newborn mice. Inactivated vaccine was prepared from the brains after inactivation with formalin and then purification by ultracentrifugation. The antigenic potency of this vaccine in vitro was determined by antibody-bound enzyme-linked immunosorbent assay (ELISA) and serial diluted vaccine bound to an aluminium hydroxide gel was inoculated into Balb/c mice to test immunogenicity. After two injections of this vaccine preparation, antibodies were detected in the mice by immunofluorescent, neutralizing and haemagglutination inhibition antibody tests. When mice immunized with this vaccine were challenged with B-1 virus and Hantaan virus (KHF-83-61BL strain), the virus titres in their lungs and spleens were significantly less than those in non-immunized mice. These results suggest that inactivated B-1 virus vaccine is effective against virus challenge by homotypic (B-1 virus) and heterotypic (Hantaan virus) viruses.     

Sexual diergism in antibody response to whole virus trivalent inactivated influenza vaccine in outbred mice

An outbred mouse model was used to determine if antibody response to immunization with whole-virus trivalent inactivated influenza vaccine (TIV) differs between the sexes. The antibody response was examined one (serum titer of IgM antibodies), and three and six weeks post-immunization (serum titer of neutralizing and total IgG antibodies and IgG subclass profile). Compared with male in female mice was found (i) the more robust IgM response against all influenza strains included in TIV and (ii) more vigorous neutralizing antibody and total IgG responses against H1N1 influenza virus at both the examined time points post-immunization. The total IgG antibody response against H3N2 and B influenza viruses was comparable between female and male mice three weeks post-immunization, but significantly greater in female mice six weeks post-immunization. The neutralizing antibody response against H3N2 and B influenza viruses did not significantly differ between sexes at both the examined points post-immunization. Finally, three weeks post-immunization subclass profile of IgG specific to the influenza strains included in TIV differed between female and male mice, reflecting the lower titer of IgG1 antibodies in female ones, so that IgG2a (contributing mainly to the total IgG) to IgG1 ratio in mice of this sex was shifted toward the former. In agreement with this shift, compared with male mice, Th1/Th2 balance in female mice was shifted toward Th1, as shown by ELISPOT. Collectively, the results showed influenza virus strain-dependent sexual dimorphism in the magnitude, dynamics and characteristics of antibody response in outbred mice immunized with TIV.     

Bovine immune colostrum against 17 strains of diarrhea bacteria and in vitro and in vivo effects of its specific IgG

Bovine colostral antibodies of cows immunized with a multivalent vaccine consisting of whole cells of 17 strains of pathogenic diarrhea bacteria were generated, and the specific IgG with high activities and titres directed against these pathogens was purified using an ammonium sulfate precipitation and verified by SDS-PAGE. We demonstrate that specific IgG has a strong activity of inhibiting in vitro growth and colonization in pathogens by agglutinating with bacteria and destroying cell walls. Normal IgG purified from non-immunized bovine colostrum is incapable of eliciting the same consequences as specific IgG. Specific IgG prevents enteroinvasive Escherichia coli/Salmonella typhi-induced diarrhea and may exert an effective protection by enhancing splenic NK cell activity, elevating IL-2 level and inhibiting excessive release of TNF-alpha in mice. Thus, the specific IgG from colostral antibodies of immunized bovine can provide effective protection or therapy for multibacteria-induced diarrhea.     

Immunisation with the glycolytic enzyme enolase confers effective protection against Candida albicans infection in mice

Candida albicans is an opportunistic human fungal pathogen that continues to be a leading cause of candidal infections in immunocompromised hosts. Enolase, an important glycolytic enzyme located on the cell wall of C. albicans, was cloned, purified, and characterized by molecular cloning, affinity chromatography and Western blotting. C57BL/6J mice were immunized with recombinant enolase subcutaneously every two weeks, and the protective effect against systemic challenge evaluated by fungal burdens in target organs, titres of specific antibodies to enolase, and by levels of Th1/2 cytokines in serum. After challenge with C. albicans strains SC5314 and 3630, fungal burdens in the liver, kidney, brain, spleen and lung were significantly decreased in immunized mice. Histopathological assessment demonstrated that enolase protected the tissue structure, and decreased the infiltration of inflammatory cells. The titres of enolase-specific IgG1 and IgG2a in the immune serum reached up to 1:51200. Furthermore, opsonization with immune serum resulted in enhanced killing of both 3630 and SC5314 by murine neutrophils. Levels of IL-12 and IL-8 in the immune serum increased, whereas the concentration of the Th2 cytokine, IL-10, was significantly higher in immunized mice compared to the control group. It was concluded that recombinant enolase effectively protected mice against disseminated candidiasis, and may be a promising target for vaccination against different strains of C. albicans.     

Isotype specific ELISAs to detect antibodies against swine vesicular disease virus and their use in epidemiology

Isotype specific ELISAs to detect antibodies against swine vesicular disease, which may help to estimate the moment of infection, were developed and validated on sera from pigs experimentally infected with four different isolates of swine vesicular disease virus. Virus specific IgM antibodies could be detected from days 3-49 and occasionally up to day 91 after infection. IgG1 antibodies were first detected at day 8 and IgG2 at day 11. IgA antibodies coincided with IgG1 antibodies, but antibody titres varied widely. From the results obtained with the sera from the experimentally infected pigs, we calculated the day at which 50% of the pigs had become positive (D50). A D50 of 5, 4, 12, 12 and 24 days was calculated, respectively, for the appearance of antibodies in the virus neutralization test, the IgM, total IgG, IgG1 and IgG2 ELISA. A D50 of 49 days was calculated for the disappearance of IgM antibodies. The isotype specific ELISAs proved to be valuable tools to study the epidemiology of the disease.     

Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice

Influenza is controlled by protective titres of neutralizing antibodies, induced with the help of CD4 T-cells, and by antiviral T-cell effector function. Adjuvants are essential for the efficient vaccination of a na?ve population against avian influenza. We evaluated a range of adjuvants for their ability to enhance, in na?ve mice, protective hemagglutination inhibition (HI) titres, which represent the generally accepted correlate of protection, virus-neutralizing titres and T-cell responses to a new generation influenza vaccine produced in cell culture. The selected adjuvants include alum, calcium phosphate (CAP), MF59, the delivery system poly-(lactide co-glycolide) (PLG) and the immune potentiator CpG. MF59 was clearly the most potent single adjuvant and induced significantly enhanced, long-lasting HI and neutralizing titres and T-cell responses in comparison to all alternatives. The combination of alum, MF59, CAP or PLG with CpG generally induced slightly more potent titres. The addition of CpG to MF59 also induced a more potent Th1 cellular immune response, represented by higher IgG2a titres and the induction of a strongly enhanced IFN-gamma response in splenocytes from immunized mice. These observations have significant implications for the development of new and improved flu vaccines against pandemic and inter-pandemic influenza virus strains.     

Enzyme immunoassay for the detection of dengue IgG and IgM antibodies using infected mosquito cells as antigen

The detection of immunoglobulin (Ig)G and IgM antibodies to dengue 1 virus was studied by a simple enzyme immunoassay, in which infected cultured cells infected with dengue virus were used as antigen (EIA-ICC). Detection of anti-dengue 1 IgG by EIA-ICC was correlated with haemagglutination assays. EIA-ICC anti-dengue 1 IgM detection was less sensitive than IgM capture enzyme-linked immunosorbent assay. IgG and IgM responses in dengue 1 infection were studied by EIA-ICC, using sera collected at different intervals after onset of illness: IgM and IgG appeared on the 4th day of disease; the highest IgM mean titres were detected on the 7th day and IgM was not detected in sera obtained after the 60th day; the highest mean titres of anti-dengue 1 IgG were seen in sera obtained between 22 and 30 d after onset of illness. EIA-ICCs for 6 flaviviruses and 1 alphavirus were conducted with sera from patients infected with dengue 1, and primary and secondary infections of other flaviviruses. The results showed that anti-dengue 1 IgG detection was sensitive, and the antibodies were cross-reactive among the flaviviruses. Anti-dengue 1 IgM detected in dengue 1 patients was mostly type specific. The pattern of secondary dengue infection, i.e. the presence of IgG and a low titre or absence of IgM antibodies, was observed in the sera of 6 patients obtained in the first week after onset of illness. EIA-ICC is useful for dengue diagnosis, surveillance and sero-epidemiological studies.     

汉滩型与汉城型汉坦病毒基因重排的初步研究

目的：探讨汉坦病毒汉滩型与汉城型之间能否发生基因重排以及发生重排的频率和特点。方法：用汉坦病毒汉滩型76－118株与汉城型SR－11株混合感染VeroE6细胞，空斑形成试验挑取子代病毒克隆株，分型聚合酶链反应方法鉴定子代病毒基因型。结果：大部分（68．19％）子代病毒的基因组来自7－118株或SR－11株；2株（4．55％）两型引物扩增均为阳性；2株（4．45％）两型引物扩增均为阴性；5株（11．36％）发生了M片段重排，3株（6．82％）发生了L片段重排，未发现S片段重排；1株M片段来自双亲株，1株S片段为双倍体。结论：证实汉坦病毒I型与II型之间可以发生重排。     

Oral vaccination with inactivated influenza vaccine induces cross-protective immunity

Oral vaccination would provide an easy and safe measure to prevent infectious diseases by facilitating mass immunization. We investigated the feasibility of oral vaccination with inactivated whole influenza virus (A/PR8/34). Oral vaccination of mice induced high levels of serum IgG and IgA antibodies specific to the homologous virus (A/PR8) as well as cross reactive to heterologous (A/California/04/09) and heterosubtypic viruses (A/Philippines/2/82). IgG1 isotype antibodies were found to be induced at significantly higher levels than IgG2a antibodies. These antibodies induced by oral vaccination exhibited hemagglutination inhibition activities. High levels of both IgG and IgA antibodies were induced in vagina and lungs. Mucosal IgA antibodies were also elicited in other sites including saliva, urine, and fecal samples. Orally vaccinated mice were completely protected against challenge with homologous or heterologous viruses, and partially protected against heterosubtypic virus. Importantly, high recall antibody secreting cell (ASC) responses were induced in spleen, indicating the generation of memory B cells by oral vaccination. The present study therefore presents new findings of cross-reactive antibodies at systemic and diverse mucosal sites, recall antibody responses, and cross-protective efficacies by oral vaccination, thus supporting a proof-of-concept that oral delivery of vaccines can be developed as an effective vaccination route.     

The Rb1 fraction of ginseng elicits a balanced Th1 and Th2 immune response

Porcine parvovirus (PPV) vaccines containing different adjuvants were evaluated for inducing Th1 or Th2 type of immunity in mice. Isotypes of antigen specific antibodies and levels of cytokines in serum and in lymphocyte culture supernatants measured by ELISA and the Gyrolab Bioaffy were used to determine the polarisation of the immune response. Enumeration of cytokine secreting cells was carried out by ELISPOT assays. Vaccines containing the ginseng-fraction Rb1 induced serum-detectable amounts of IL-4 and IL-10 as early as 24h after primary injection that was confirmed in sera collected at 24 and 72 h post re-vaccination. Five weeks after booster, immune lymphocytes were still producing large amounts of cytokines including IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha and the antibody titres were still similar to those titres recorded 1 week post booster. The Rb1 adjuvanted vaccines stimulated similar titres of antigen specific IgG1, IgG(2a) and IgG(2b). Thus, the cytokine and the serological data indicated that the Rb1 fraction of ginseng elicits a balanced Th1 and Th2 immune response.