抗创伤弧菌单克隆抗体杂交瘤细胞系的建立及特性鉴定

目的:制备高效、特异的抗创伤弧菌的单克隆抗体(mAb),并进行特性鉴定。方法:用创伤弧菌菌体蛋白抗原免疫小鼠,利用杂交瘤细胞融合技术,制备抗创伤弧菌的杂交瘤细胞株。用ELISA法及Western blot等筛选、鉴定其与创伤弧菌溶血素蛋白(vvhA)及其他重要海洋细菌的交叉反应性和效价。结果:共获得5株抗创伤弧菌的mAb,鉴定结果表明,5株mAb均具有良好的特异性和免疫反应性。结论:获得5株抗创伤弧菌的特异性mAb,为建立创伤弧菌快速检测试剂盒提供了重要制剂。     

副溶血弧菌单克隆抗体的制备和鉴定

目的:制备副溶血弧菌(Vibrio parahaemolyticus)单克隆抗体(mAb)并进行鉴定。方法:用灭活的副溶血弧菌免疫BALB/c小鼠,采用常规杂交瘤技术制备副溶血弧菌mAb。用间接ELISA法对mAb的效价进行测定。结果:获得4株能稳定分泌副溶血弧菌mAb的杂交瘤细胞株,分别命名为1E3、5F12、5D8、6H9。经测定杂交瘤细胞腹水mAb效价分别为1×10-4～1×10-7。4株杂交瘤细胞系所分泌的mAb亚类分别是:IgG2b,IgG2a,IgG2b,IgG3。交叉反应试验显示,mAb 5D8与弧菌属其他几种细菌均不起反应,特异性强。副溶血弧菌生长抑制及动物保护实验结果表明4株mAb均能不同程度地抑制副溶血弧菌的生长并对动物具有保护作用,其中5F12保护效果最强。结论:成功地制备能稳定分泌副溶血弧菌mAb的杂交瘤细胞株,为建立该病的免疫学诊断及防治的奠定了基础。     

豚鼠气单胞菌抗独特型单克隆抗体的制备和初步鉴定

目的:制备豚鼠气单胞菌(Aeromonascaviae)抗独特型单克隆抗体(AIdmAb)并进行鉴定。方法:以具有中和作用的抗豚鼠气单胞菌mAb1F1免疫BALB/c小鼠,采用常规杂交瘤技术制备豚鼠气单胞菌AIdmAb。用ELISA法对mAb的效价及其模拟豚鼠气单胞菌的特性进行鉴定。结果:获得4株能稳定分泌豚鼠气单胞菌AIdmAb的杂交瘤细胞株,分别命名为1E12、4F6、6G6、7C1。经测定杂交瘤细胞腹水mAb效价为10-3～10-4。竞争抑制实验结果表明4株AIdmAb均能不同程度地抑制豚鼠气单胞菌与兔抗豚鼠气单胞菌多克隆抗体的结合。制备杂交瘤细胞腹水的BALB/c小鼠血清均能与豚鼠气单胞菌反应,其效价为1∶50～1∶800。结论:成功地制备了豚鼠气单胞菌抗独特型mAb的杂交瘤细胞株,为该菌的抗独特型疫苗制备研究奠定了基础。     

抗温和气单胞菌单克隆抗体的制备及初步鉴定

目的制备抗温和气单胞菌(Aeromonassobria,As)单克隆抗体(mAb)并进行鉴定。方法以灭活的温和气单胞菌免疫BALB/c小鼠,采用常规杂交瘤技术制备抗温和气单胞菌mAb。用间接ELISA法对mAb的特性进行初步鉴定。结果获得6株能稳定分泌抗温和气单胞菌mAb的杂交瘤细胞株,分别命名为1A8、2A8、2F11、3C6、3D11、4G2。经测定杂交瘤细胞培养上清及其诱生的腹水mAb效价分别为1×10-2~1×10-3、1×10-5~1×10-6。6株杂交瘤细胞系所分泌的mAb亚类是2A8、2F11为IgG1,1A8、3D11为IgG2a,3C6为IgG2b,4G2为IgG3。其中mAb3C6经交叉反应试验证明,与弧菌属其他几种细菌均不起反应,特异性强。结论成功地制备了抗温和气单胞菌mAb的杂交瘤细胞株,为该病的免疫学诊断及防治提供了基础。     

抗豚鼠气单胞菌单克隆抗体杂交瘤细胞株的建立及其特性鉴定

目的:制备抗豚鼠气单胞菌(Aeromonascaviae)单克隆抗体(mAb)并进行鉴定。方法:用灭活的豚鼠气单胞菌全菌免疫BALB/c小鼠,采用杂交瘤技术制备mAb。用间接ELISA法对mAb的效价及其与其他细菌的交叉反应性进行鉴定;用免疫组化染色法对mAb的免疫反应性进行鉴定。结果:获得7株能稳定分泌抗豚鼠气单胞菌mAb的杂交瘤细胞株,分别命名为0E10、1A2、1C4、1F1、1F10、1H8和2A1。经测定杂交瘤细胞培养上清及腹水mAb的效价,分别为10-1~10-2和10-3~10-5。mAb的Ig亚类鉴定表明,1F1为IgG1,1C4、1F10和1H8为IgG2a,2A1为IgG2b,0E10和1A2为IgG3。交叉反应试验显示,各株mAb均具有较好的特异性。免疫组化染色结果表明,mAb能和豚鼠气单胞菌特异性结合。结论:成功地制备分泌抗豚鼠气单胞菌mAb的杂交瘤细胞株,为该菌的快速检测及免疫学研究奠定了基础。     

嗜水气单胞菌抗独特型单克隆抗体的制备和初步鉴定

目的:制备嗜水气单胞菌(Aeromonas hydophila)抗独特型单克隆抗体(AId mAb)并进行鉴定.方法:以具有中和作用的抗嗜水气单胞菌 mAb 1G10免疫 BALB/c小鼠,采用常规杂交瘤技术制备嗜水气单胞菌 AId mAb.用 ELISA法对 mAb的效价及其模拟嗜水气单胞菌的特性进行鉴定.结果:获得4株能稳定分泌嗜水气单胞菌 AId mAb的杂交瘤细胞株,分别命名为1F9、 1H10、 3F5、 4G3.经测定杂交瘤细胞腹水 mAb效价为10 -4～10 -5.竞争抑制实验结果表明 4株 AId mAb均能不同程度地抑制嗜水气单胞菌与兔抗嗜水气单胞菌多克隆抗体结合,其中两株 1F9、 1H10具有较强的抑制作用.用作制备抗独特性抗体腹水的小鼠,其血清均能检测与嗜水气单胞菌进行免疫结合的抗体,经 ELISA检测,其效价为 1: 100 ～1: 1 000.结论:成功地制备了产生嗜水气单胞菌抗独特型抗体的杂交瘤细胞株,为制备该菌的抗独特性抗体疫苗奠定了基础.     

抗SARS冠状病毒M蛋白片段单克隆抗体的制备与初步鉴定

目的:制备抗SARS冠状病毒(SARS-CoV)M蛋白N端1~43氨基酸(aa)单克隆抗体(mAb),并对其特性进行初步鉴定。方法:用纯化的SARS-M/GST融合蛋白抗原免疫BALB/c小鼠,采用杂交瘤技术制备抗M蛋白片段的mAb。用间接ELISA法筛选能分泌抗M蛋白片段mAb的杂交瘤细胞株。Westernblot和间接ELISA鉴定所获mAb的特异性,并用小鼠mAb亚类测定试剂盒检测所获的mAbIg亚类。为分析mAb识别位点,进一步将M蛋白片段截短为部分重叠的2段表达,以Westernblot初步定位mAb识别位点。结果:获得1株可分泌特异性抗SARS-CoVM蛋白片段的mAb杂交瘤细胞株(3H9),Ig亚类鉴定为IgG2a,轻链为κ型。Westernblot显示其mAb可特异识别SARS-CoVM蛋白N端1~43aa,间接ELISA证实mAb可与包被于聚苯乙烯微孔板上的SARS病毒全蛋白抗原发生特异性反应,其识别位点位于M蛋白N端16~28aa。结论:成功获得1株抗SARS-CoVM蛋白N端1~43aamAb杂交瘤细胞,并初步定位其识别位点。     

两株鼠抗人PD-1单克隆抗体的制备及其生物学特性鉴定

目的: 制备鼠抗人PD-1单克隆抗体(mAb)及鉴定其生物学特性.方法: 以基因转染细胞PD-1/L929为免疫原, 免疫BALB/c小鼠;采用淋巴细胞杂交瘤技术进行细胞融合, 经选择性克隆化培养及流式细胞术(FCM)分析, 筛选分泌鼠抗人PD-1分子mAb的杂交瘤细胞株;采用Ig亚型快速定性试纸法、染色体核型分析、 Western blot、竞争结合抑制试验及肿瘤细胞株测定等方法对mAb进行生物学特性鉴定.结果: 获得了2株持续稳定分泌鼠抗人PD-1mAb的杂交瘤细胞株, 命名为1F2和5F10.对其生物学特性的鉴定结果表明, 均为条带相对分子质量在(Mr)55 000左右的免疫球蛋白, 具有不同的PD-1结合位点;1F2 mAb可识别SKHep-1和7721细胞株表面的PD-1分子, 而5F10可识别Raji细胞株的PD-1分子.结论: 成功获得了2株鼠抗人PD-1杂交瘤及其分泌的mAb.     

His标签单克隆抗体的制备、鉴定及初步应用

目的:制备和鉴定抗His标签单克隆抗体(mAb),初步建立检测和纯化带His标签融合蛋白的方法.方法:用碳化二亚胺法合成His-tag完全抗原,免疫BALB/c小鼠,采用杂交瘤技术进行细胞融合,通过有限稀释法和间接ELISA法克隆和筛选阳性杂交瘤细胞株.常规制备腹水,用辛酸-硫酸铵法纯化,并对纯化的mAb进行特异性鉴定.结果:His-tag完全抗原偶联成功,经细胞融合、筛选及克隆化,成功获得1株分泌His标签mAb的杂交瘤细胞株.制备腹水测得腹水效价高于 1:106,且此株mAb与其他融合蛋白标签无交叉反应,并在含His标签融合蛋白的鉴定实验中取得了满意效果.结论:成功制备了1株His标签mAb,为带His标签融合蛋白的研究应用提供了重要的工具.     

血管内皮生长因子(VEGF)单克隆抗体的制备及鉴定

目的:制备血管内皮生长因子(VEGF)单克隆抗体(mAb),并对其特性进行鉴定.方法:以重组His-Trx-VEGF1-165蛋白为抗原,通过免疫、融合、克隆和筛选等方法制备VEGF mAb,制备小鼠腹水并测定其效价、染色体数目及免疫球蛋白亚类,用Western blot方法分析其特异性.结果:成功筛选出能稳定分泌的VEGF mAb杂交瘤细胞株4E4-H5,制备腹水的ELISA效价为1:106;染色体数目为105条;抗体分型为IgG1亚型,κ轻链;Western blot证实所获得的mAb可与VEGF全长蛋白发生特异性反应,但是与融合蛋白的标签蛋白His-Trx不发生反应.结论:制备的杂交瘤细胞株能稳定分泌高滴度和高特异性的VEGF mAb,为建立能够用于肝癌诊断的血清VEGF检测技术提供了参考.     

Rapid detection ofVibrio cholerae O∶1 by motility inhibition and immunofluorescence with monoclonal antibodies

Monoclonal antibodies against the group and type specific antigens ofVibrio cholerae O∶1 lipopolysaccharide were used for the rapid detection ofVibrio cholerae strains by motility inhibition and immunofluorescence. Motility inhibition of liveVibrio cholerae O:1 was obtained with group specific monoclonal antibodies. Monoclonal antibodies against the type specific antigens B (Ogawa) and C (Inaba) inhibited motility of strains of homologous serotypes only. Indirect immunofluorescence of heatfixed bacteria with monoclonal antibodies and fluorescein-isothiocyanate conjugated rabbit anti-mouse immunoglobulin was also shown to be suitable for the rapid detection ofVibrio cholerae O∶1. Both tests were highly specific and no cross-reactions were observed with strains of non-O∶1 vibrios,Escherichia coli orSalmonella spp. tested. However, a weak fluorescence of some Ogawa strains was observed when high concentrations of Inaba specific monoclonal antibodies were used.     

Monoclonal antibodies against group- and type-specific lipopolysaccharide antigens of Vibrio cholerae O:1

Hybrid cell lines producing monoclonal antibodies against the O-antigenic determinants of Vibrio cholerae O:1 have been established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay inhibition experiments by using lipopolysaccharides from V. cholerae O:1 strains and type strains of groups O:2 and O:21. The anti-A antibody was of the immunoglobulin M (IgM) class, whereas the anti-B and -C antibodies were IgG3. The antibodies had a good agglutinating capacity when tested against V. cholerae O:1 strains in the slide agglutination test.     

Molecular Cloning of a Bacteroides caccae TonB-Linked Outer Membrane Protein Identified by an Inflammatory Bowel Disease Marker Antibody

Commensal enteric bacteria are a required pathogenic factor in inflammatory bowel disease (IBD), but the identity of the pertinent bacterial species is unresolved. Using an IBD-associated pANCA monoclonal antibody, a 100-kDa protein was recently characterized from an IBD clinical isolate of Bacteroides caccae (p2Lc3). In this study, consensus oligonucleotides were designed from 100-kDa peptides and used to identify a single-copy gene from the p2Lc3 genome. Sequence analysis of the genomic clone revealed a 2,844-bp (948 amino acid) open reading frame encoding features typical of the TonB-linked outer membrane protein family. This gene, termed ompW, was detected by Southern analysis only in B. caccae and was absent in other species of Bacteroides and gram-negative coliforms. The closest homologues of OmpW included the outer membrane proteins SusC of Bacteroides thetaiotaomicron and RagA of Porphyromonas gingivalis. Recombinant OmpW protein was immunoreactive with the monoclonal antibody, and serum anti-OmpW immunoglobulin A levels were elevated in a Crohn's disease patient subset. These findings suggest that OmpW may be a target of the IBD-associated immune response and reveal its structural relationship to a bacterial virulence factor of P. gingivalis and periodontal disease.     

Monoclonal antibodies against Vibrio cholerae lipopolysaccharide.

A cell line producing monoclonal antibodies directed against the core region of Vibrio cholerae lipopolysaccharide has been established. These antibodies were inhibited by lipopolysaccharide preparations of both O-group 1 vibrios and some non-O-group 1 vibrios as detected in enzyme-linked immunosorbent assay-inhibition experiments. Coagglutination experiments with monoclonal and polyclonal antibodies adsorbed to protein A-carrying staphylococci were performed. All V. cholerae strains tested, regardless of serotype, were agglutinated when mixed with staphylococci coated with the monoclonal antibodies, whereas staphylococci coated with group-specific (O1) polyclonal antibodies only agglutinated with O-group 1 vibrios.     

Differential expression of the outer membrane protein W (OmpW) stress response in enterohemorrhagic Escherichia coli O157:H7 corresponds to the viable but non-culturable state

During an outbreak of enterohemorrhagic Escherichia coli (EHEC) O157, we showed previously that food isolates were resistant to oxidative stress, while patient isolates were sensitive to it. Because food isolates increased stress-sensitivity after mouse passage, this change most likely occurred during passage through patients. Here we demonstrate that the phenotypic change occurring during mouse passage correlates with the stress response of outer membrane protein W (OmpW) in EHEC O157 strains. Upon induction of oxidative stress, OmpW was highly expressed only in the stress-sensitive MP37 strain, obtained by mouse passage of food strain F2, but not in the F2 strain. Western blotting confirmed that expression of OmpW was induced in the viable but non-culturable (VBNC) state. Deletion of ompW in the MP37 strain increased recovery from dormancy, while overexpression of OmpW in the F2 strain decreased recovery when exposed to oxidative stress, suggesting that high levels of OmpW sensitize the bacteria to stress. DNA alignment revealed that the class I integron (int1I) fragments flanking the ompW gene are oriented in opposite directions between stress-resistant and -sensitive strains. All stress-sensitive strains induced ompW under stress. We propose that the different stress response of OmpW was introduced by genetic alteration during in vivo passage.     

Elevated Serum Anti-Saccharomyces cerevisiae, Anti-I2 and Anti-OmpW Antibody Levels in Patients with Suspicion of Celiac Disease

OBJECTIVES: Expression of anti-Saccharomyces cerevisiae antibodies (ASCA) identifies patients and individuals at risk for Crohn's disease and has also been reported in 40-60% of celiac disease (CD) cases, suggesting a role of host response to enteric microbiota in the development of inflammatory lesions. In this prospective study in patients with suspicion of CD, we evaluate the frequency and association of ASCA to serological responses for other host microbial targets formally associated with Crohn's disease, including the Pseudomonas fluorescens associated sequence I2 and a Bacteroides caccae TonB-linked outer membrane protein, OmpW. METHODS: Small bowel mucosal biopsies were taken from 242 patients with suspicion of CD, their sera were tested for antibodies to tissue transglutaminase (tTG), ASCA, I2, and OmpW. Eighty adult healthy blood donors were used as controls. RESULTS: The diagnosis of CD was confirmed on biopsy in 134 cases. The occurrence of ASCA and I2 positivity was significantly higher in adult CD patients than in patients with non-CD disease. Anti-I2 levels in the sera were significantly higher in adult CD patients than in non-CD disease or the controls and anti-OmpW levels in CD and non-CD patients when compared to controls. Positive seroreactivity to OmpW seemed to increase with age. Of the CD patients, 90% were seropositive for at least one microbial antigen tested. CONCLUSIONS: This study demonstrates a mosaic of disease-related serological responses to microbial antigens in patients with CD. Immune responses to commensal enteric bacteria may play a role in the small intestine mucosal damage in CD.     

O1群小川型霍乱弧菌单克隆抗体的制备与鉴定

目的制备抗O1群小川型霍乱弧菌(VibriocholeraeO1serotypeOgawa)特异性单克隆抗体(McAb),为霍乱的早期快速诊断提供有力的抗体工具。方法以灭活的O1群小川型霍乱弧菌免疫Balbc小鼠,通过杂交瘤技术制备针对O1群小川型霍乱弧菌的McAb,以间接ELISA法对所需的杂交瘤细胞株进行筛选,分析其亚类,检测其效价及相对亲和力,以间接ELISA和Westernblot鉴定McAb特异性,并进行McAb结合表位分析。结果融合了602株能分泌抗O1群小川型霍乱弧菌McAb的杂交瘤细胞株,最后得到5株能稳定分泌特异性的针对该McAb的细胞株,其抗体亚类分别为3株IgG1,1株IgG2b,1株IgG3;腹水效价均达1×10-6;亲和常数在1×108～1×109之间。间接ELISA法及Westernblot证实所获的McAb可与O1群小川型霍乱弧菌发生特异性反应。ELISA相加实验结果显示除有2株McAb识别相同的抗原表位外,其余均识别不同的抗原表位。结论获得霍乱弧菌O1群小川型特异性McAb,为O1群小川型霍乱早期快速诊断和发病机理的研究提供基础。     

The specificity of Atlantic salmon antibodies made against the fish pathogen Vibrio salmonicida, establishing the surface protein VS-P1 as the dominating antigen

The specificity of salmon (Salmo salar) antibodies made against the fish pathogen Vibrio salmonicida was studied. Salmon immunized with V. salmonicida emulisified in Freund's complete adjuvant produced antibodies which preferentially bound to a 40 KD outer surface molecule (VS-P1). Moreover, the bulk of the antibodies were specific for one particular epitope on VS-P1, defined by a mouse monoclonal antibody - as detected with a blocking ELISA. The data imply that salmon B cells mainly (90%) recognize one particular determinant on V. salmonicida and thus express a limited repertoire of antibody specificities against this pathogen.     

Monoclonal antibody-based enzyme-linked immunosorbent assays for identification and serotyping of Vibrio cholerae O1.

Monoclonal antibodies directed against O-specific antigens of Vibrio cholerae O1 lipopolysaccharide were used in two different enzyme-linked immunosorbent assays (ELISAs), designed for identification and serotyping of V. cholerae O1. In the sandwich ELISA, a monoclonal antibody against the group-specific antigen was used as capture antibody, whereas peroxidase-conjugated monoclonal antibodies directed against group- and type-specific antigens were used as the second antibodies. Monoclonal antibodies were also used in ELISA inhibition tests with whole bacteria as inhibitors in microtiter trays coated with V. cholerae O1 lipopolysaccharide. In addition, the monoclonal antibodies were shown to be useful in slide agglutination tests. The enzyme immunoassays were equally sensitive, showing positive reactions with all V. cholerae O1 strains tested, whereas all V. cholerae non-O1 as well as strains of Escherichia coli, Shigella sonnei, Salmonella spp., Citrobacter freundii, and Brucella abortus were negative. The microtiter application makes the immunoassays suitable with low consumption of reagents for screening of samples from suspected cases as well as from the environment.