（一）猪鼻支原体免疫胶体金快速检测卡的研制 （二）抗念珠状链杆菌单克隆抗体的制备和鉴定
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摘要
胶体金免疫层析检测(gold immunochromatography assay，GICA)，以其特异、敏感、快速的优点，广泛应用于病原微生物的流行病学调查和感染性疾病的临床诊断。本研究根据猪鼻支原体感染在人体血清抗体效价不高，其病原体在人体腔道寄生的特点，确定双抗体夹心法诊断抗原的GICA检测模式。以特异性和敏感性较高的配对单克隆抗体(McAb)作为诊断试剂，然后进行条件筛选和优化建立GICA检测卡(GICAs)，结果如下：
首先，层析材料的制备和选择。采用柠檬酸三钠还原法制备胶体金颗粒，调节还原剂的用量可得到不同粒径的胶体金，可见光光谱扫描鉴定表明颗粒均匀，粒径大小符合理论值。选择合适粒径的胶体金标记McAb，即为金标抗体，另一株McAb为包被抗体；对诊断试剂和层析材料通过层析性能和应用效果两方面进行筛选，确定缓冲液环境，抗体工作浓度等。
其次，GICA的建立和在夹心模式下的整体优化实验，降低检测限，减少非特异性反应，提高稳定性。
最后，GICA应用的初步评价。检测猪鼻支原体4d的液体培养物，其检测限为1.72×10~3CFU／mL，检测全菌蛋白检测限为50ng／100μL；特异性试验表明与实验动物和人易感11种细菌和11种支原体均无交叉反应。阳性结果可在5min内判定，弱阳性结果需时约10min。批内重复性和批间重复性好，稳定性试验结果表明其4℃保存6个月以上在检测限以上仍能得到清晰的阳性结果。
本文研制的GICAs检测猪鼻支原体特异性强、灵敏度高，操作简单、结果快速，可用于感染的流行病学调查和临床诊断。
念珠状链杆菌(Streptobacillus moniliformis，以下简称SM)为人鼠共患病病原。目前国内外尚缺乏针对SM的快速检测试剂盒，传统的分离培养法需时较长且该菌易形成L形变异，PCR方法检测步骤较为繁琐且容易受条件限制，均不适用于实验动物隐性感染和临床感染病例的快速诊断。本文以经典的细胞融合技术制备抗SM单克隆抗体，为进一步建立以血清学诊断试剂为基础的快速检测方法奠定基础。
首先，抗SM单克隆抗体的制备。以SM全菌蛋白为免疫原，免疫BALB／c小鼠，经过基础免疫、加强免疫3次以上，待免疫血清抗体效价达到1：10~5以上后进行冲击免疫，3天后取脾细胞，与处于对数生长期的SP2／0细胞融合，以甲基纤维素半固体法进行克隆，亚克隆3次，以间接ELISA方法对克隆后的细胞株进行筛选，最后共筛选出12株分泌抗体阳性的融合细胞株。
其次，抗SM单克隆抗体的鉴定。通过染色体计数试验证明12株细胞皆为杂交瘤细胞；亚类测定证实其分属于IgG_1、IgG_(2a)、IgG_(2b)以及IgM类；Westem-blot实验表明单抗5G4结合的抗原分子量为32×10~3，6D10结合的抗原分子量为66×10~3，589结合的抗原分子量有2个条带，在60-67×10~3之间，为SM种特异性抗原；IFA结果显示，单抗5G4能够与SM表面抗原结合；ELISA实验初步表明单抗5G4与实验动物常见病原菌无交叉反应，提示单抗5G4有可能作为诊断抗体直接检测抗原或者作为诊断抗原纯化的亲和层析配体。在后续SM血清学诊断方法的研究中，我们需要对其敏感性和特异性以及存在的问题(混和亚类)作进一步的研究。
Gold immunochromatography assay (GICA) has been used widely in epidemiology investigations and clinical diagnosis especially infection diseases because of its specificity, sensitivity, convenience and rapidness. The aim of this study is to establish sandwich GICAs to detect M. hyorhinis due to low level antibody in serum and the fact that they exist in human's cavity. The most suitable double antibody was selected to be used in sandwich GICAs. The results are as follow:
Firstly, GICA materials has been prepared and screened primarily. Colloidal gold was prepared by reduction of HAuCl_4 with trisodium citrate. Different size Colloidal gold particles have been prepared by using different volumes of trisodium citrate. Their diameters and particle distribution uniformity were detected through visible spectroscopy. One of them was selected to be labeled with one of the double antibody, another was used as coat antibody. All the GICA materials were screened based on their chromatography ability and application value, the buffers and the work concentration of antibodies were also determined in methodology.
Further more, the qualifications and conditions of sandwich GICA were selected in the second time and they were optimized for the development of sensitivity, specificity and stability.
Finally, the applications of the GICAs were evaluated in sensitivity, specificity and stability. Threshold value of the GICAs was 1.72×10~3CFU/mL and 500ng/100μL of bacterial protein, without cross reaction with 11 bacteria and 11 mycoplasmas which are common in human and laboratory animals. The process would take about 5 minutes for positive result and 10 minutes for lightly positive result. Positive result could be repeated using either same or different groups of the GICAs. The positive result could be seen as before above the threshold value even the GICAs were stored at 4℃more than six months later.
In a word, we have successfully developed the GICAs firstly that can be used for epidemiology studies and clinic diagnosis of M. hyorhinis infections for its excellence in sensitivity, specificity and stability.
Streptobacillus moniliformis (SM) is the pathogenic bacteria both in human and rat. At present it is absent of rapid serological reagent to detect the disease caused by SM. the conventional culture method is time costing and the bacteria is easy to be induced to L-form, PCR is cockamamie and be limited. So we prepared the monoclonal antibodies (McAb) anti SM using routine cell fusion technique for the further development of rapid serodiagnostic reagent to detect SM.
Firstly, the hybridoma cell strains was prepared as follows: Titers of ascetic fluid were up to 1: 10~5 by immunizing BALB/c mice with bacteria protein for one basic and 3 trengthen immunity, then get the spleen cells and confuse them with the SP2/0 cell strain. The cells were Clone Repeat 3 times using methyl cellulose semisolid culture and 12 antibody-positive strains were screened finally by ELISA.
Secondly, the McAb is identified as follows: The 12 hybridoma cell strains were confirmed by chromosome count. The McAb' subtypes were belong to IgG1、IgG2a、IgG2b and IgM by ELISA. McAb 5G4 had a clear reactivity to the bacteria membrane antigen by IFA, their specificity was primarily tested by ELISA. The Western-blot shows that the molecular weight of the antigens that the McAb 5G4、6D10、5B9 recognized were located at 32×10~3、66×10~3 and 60-67×10~3 and they are common strains of SM of different origin, which indicates that they may be used in serodiagnostic reagent as the antibodies anti genus antigen of SM. But their sensitivity and some problems such as mix subtypes of Ig must be researched furthermore when used as serodiagnostic reagent.
首先，层析材料的制备和选择。采用柠檬酸三钠还原法制备胶体金颗粒，调节还原剂的用量可得到不同粒径的胶体金，可见光光谱扫描鉴定表明颗粒均匀，粒径大小符合理论值。选择合适粒径的胶体金标记McAb，即为金标抗体，另一株McAb为包被抗体；对诊断试剂和层析材料通过层析性能和应用效果两方面进行筛选，确定缓冲液环境，抗体工作浓度等。
其次，GICA的建立和在夹心模式下的整体优化实验，降低检测限，减少非特异性反应，提高稳定性。
最后，GICA应用的初步评价。检测猪鼻支原体4d的液体培养物，其检测限为1.72×10~3CFU／mL，检测全菌蛋白检测限为50ng／100μL；特异性试验表明与实验动物和人易感11种细菌和11种支原体均无交叉反应。阳性结果可在5min内判定，弱阳性结果需时约10min。批内重复性和批间重复性好，稳定性试验结果表明其4℃保存6个月以上在检测限以上仍能得到清晰的阳性结果。
本文研制的GICAs检测猪鼻支原体特异性强、灵敏度高，操作简单、结果快速，可用于感染的流行病学调查和临床诊断。
念珠状链杆菌(Streptobacillus moniliformis，以下简称SM)为人鼠共患病病原。目前国内外尚缺乏针对SM的快速检测试剂盒，传统的分离培养法需时较长且该菌易形成L形变异，PCR方法检测步骤较为繁琐且容易受条件限制，均不适用于实验动物隐性感染和临床感染病例的快速诊断。本文以经典的细胞融合技术制备抗SM单克隆抗体，为进一步建立以血清学诊断试剂为基础的快速检测方法奠定基础。
首先，抗SM单克隆抗体的制备。以SM全菌蛋白为免疫原，免疫BALB／c小鼠，经过基础免疫、加强免疫3次以上，待免疫血清抗体效价达到1：10~5以上后进行冲击免疫，3天后取脾细胞，与处于对数生长期的SP2／0细胞融合，以甲基纤维素半固体法进行克隆，亚克隆3次，以间接ELISA方法对克隆后的细胞株进行筛选，最后共筛选出12株分泌抗体阳性的融合细胞株。
其次，抗SM单克隆抗体的鉴定。通过染色体计数试验证明12株细胞皆为杂交瘤细胞；亚类测定证实其分属于IgG_1、IgG_(2a)、IgG_(2b)以及IgM类；Westem-blot实验表明单抗5G4结合的抗原分子量为32×10~3，6D10结合的抗原分子量为66×10~3，589结合的抗原分子量有2个条带，在60-67×10~3之间，为SM种特异性抗原；IFA结果显示，单抗5G4能够与SM表面抗原结合；ELISA实验初步表明单抗5G4与实验动物常见病原菌无交叉反应，提示单抗5G4有可能作为诊断抗体直接检测抗原或者作为诊断抗原纯化的亲和层析配体。在后续SM血清学诊断方法的研究中，我们需要对其敏感性和特异性以及存在的问题(混和亚类)作进一步的研究。
Gold immunochromatography assay (GICA) has been used widely in epidemiology investigations and clinical diagnosis especially infection diseases because of its specificity, sensitivity, convenience and rapidness. The aim of this study is to establish sandwich GICAs to detect M. hyorhinis due to low level antibody in serum and the fact that they exist in human's cavity. The most suitable double antibody was selected to be used in sandwich GICAs. The results are as follow:
Firstly, GICA materials has been prepared and screened primarily. Colloidal gold was prepared by reduction of HAuCl_4 with trisodium citrate. Different size Colloidal gold particles have been prepared by using different volumes of trisodium citrate. Their diameters and particle distribution uniformity were detected through visible spectroscopy. One of them was selected to be labeled with one of the double antibody, another was used as coat antibody. All the GICA materials were screened based on their chromatography ability and application value, the buffers and the work concentration of antibodies were also determined in methodology.
Further more, the qualifications and conditions of sandwich GICA were selected in the second time and they were optimized for the development of sensitivity, specificity and stability.
Finally, the applications of the GICAs were evaluated in sensitivity, specificity and stability. Threshold value of the GICAs was 1.72×10~3CFU/mL and 500ng/100μL of bacterial protein, without cross reaction with 11 bacteria and 11 mycoplasmas which are common in human and laboratory animals. The process would take about 5 minutes for positive result and 10 minutes for lightly positive result. Positive result could be repeated using either same or different groups of the GICAs. The positive result could be seen as before above the threshold value even the GICAs were stored at 4℃more than six months later.
In a word, we have successfully developed the GICAs firstly that can be used for epidemiology studies and clinic diagnosis of M. hyorhinis infections for its excellence in sensitivity, specificity and stability.
Streptobacillus moniliformis (SM) is the pathogenic bacteria both in human and rat. At present it is absent of rapid serological reagent to detect the disease caused by SM. the conventional culture method is time costing and the bacteria is easy to be induced to L-form, PCR is cockamamie and be limited. So we prepared the monoclonal antibodies (McAb) anti SM using routine cell fusion technique for the further development of rapid serodiagnostic reagent to detect SM.
Firstly, the hybridoma cell strains was prepared as follows: Titers of ascetic fluid were up to 1: 10~5 by immunizing BALB/c mice with bacteria protein for one basic and 3 trengthen immunity, then get the spleen cells and confuse them with the SP2/0 cell strain. The cells were Clone Repeat 3 times using methyl cellulose semisolid culture and 12 antibody-positive strains were screened finally by ELISA.
Secondly, the McAb is identified as follows: The 12 hybridoma cell strains were confirmed by chromosome count. The McAb' subtypes were belong to IgG1、IgG2a、IgG2b and IgM by ELISA. McAb 5G4 had a clear reactivity to the bacteria membrane antigen by IFA, their specificity was primarily tested by ELISA. The Western-blot shows that the molecular weight of the antigens that the McAb 5G4、6D10、5B9 recognized were located at 32×10~3、66×10~3 and 60-67×10~3 and they are common strains of SM of different origin, which indicates that they may be used in serodiagnostic reagent as the antibodies anti genus antigen of SM. But their sensitivity and some problems such as mix subtypes of Ig must be researched furthermore when used as serodiagnostic reagent.
引文
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