金黄色葡萄球菌A型肠毒素单克隆抗体的制备及ELISA检测方法的建立
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摘要
金黄色葡萄球菌是引起化脓性疾病的重要病原菌，其产生的肠毒素则是世界范围内引起食物中毒的重要原因之一。其中以A型肠毒素引起食物中毒的发生率最高，因此，建立敏感、快速的检测金黄色葡萄球菌A型肠毒素(Staphylococcal aureusenterotoxins A，简称SEA)的方法，是进行食品安全检测，预防食物中毒的必要条件，也有助于金黄色葡萄球菌感染所致化脓性疾病的早期诊断，具有重要的临床意义和卫生意义。抗SEA单克隆抗体的应用，能够有效地提高检测的灵敏度和特异性。目前，国内市场上还不能提供有效的抗SEA单克隆抗体。因此，为了适应临床检测和基础研究的需要，本研究制备了抗SEA单克隆抗体，并在此基础上建立了检测食品中金黄色葡萄球菌A型肠毒素的间接竞争ELISA方法。
本试验用SEA经多次免疫BALB／c小鼠后，取其脾细胞与SP2／0骨髓瘤细胞在50％PEG的作用下进行融合。用间接ELISA方法初步筛选出能分泌抗SEA单克隆抗体的杂交瘤细胞，用有限稀释法进行克隆化，直到克隆后的细胞孔达到100％阳性，即筛选到了只分泌一种抗体的杂交瘤细胞。本试验共筛选到三株杂交瘤细胞株，分别命名为3C1、3G10和2A10。对其进行鉴定，结果如下：在经4个月的连续传代培养和冻存6个月后复苏检测，其分泌抗体的能力差异不显著，表明其稳定性好；其染色体平均数目在87～98条之间；经亚类鉴定，三株杂交瘤细胞分泌的抗体类型均为IgG1亚类，轻链类型均为k链；ELISA交叉试验和Western-blot分析结果表明，三株抗金黄色葡萄球菌A型肠毒素的单克隆抗体与B型肠毒素、C1型肠毒素无任何交叉反应，且能特异性地与A型肠毒素结合；经抗原表位特异性分析，三株单克隆抗体的识别表位相同或相近；其相对亲和力的测定结果是3C1＞3G10＞2A10；抗体的效价水平为3C1＞3G10＞2A10，其中3C1株杂交瘤细胞的腹水效价达1∶819 200。以3C1杂交瘤细胞株的腹水型抗体为原料，建立了检测金黄色葡萄球菌A型肠毒素的间接竞争ELISA方法。通过对试验条件的系列优化，最终确定其反应条件如下：包被浓度为0.8μg／mL，包被条件为37℃2h；封闭条件为0.5％BSA封闭液37℃1h；抗体工作浓度为1∶9 600，样品和抗体的加样比为30∶70，竞争时间为37℃1h；酶标二抗浓度为1∶6000，反应时间为37℃30min；显色条件为37℃避光反应10min。在该反应条件下，获得一条标准曲线，其回归方程为y=-0.2902x+0.8338，相关系数r=0.9952，检测限为0.7311ng／mL，能满足目前对食品中肠毒素的检测要求。批内和批间变异系数分别为，1.22％～13.47％和9.31％～13.47％，具有较好的重复性和精确度；与B型和C1型肠毒素没有任何交叉反应，表明该方法的特异性强。用建立的检测A型肠毒素间接竞争ELISA方法对肉制品中添加的SEA进行检测。其定量限为0.8557ng／mL，平均回收率在60～120％之间，批内和批间变异系数均＜20％，说明本试验建立的检测金黄色葡萄球菌A型肠毒素间接竞争ELISA方法可用于临床检测，为金黄色葡萄球菌A型肠毒素检测试剂盒的研制奠定了基础，对预防葡萄球菌性食物中毒具有积极作用。
Staphylococcus aureus is a common pathogen that causes suppurative disease, and can produce staphylococcus enterotoxins, which are a leading reasons of food poisoning worldwide. Among all of the food poisoning caused by enterotoxins, staphylococcus enterotoxin type A (SEA) is the most important. Therefore, establishing a sensitive, rapid detection assay to SEA is necessary for food-safety, also contributing to early diagnosis of suppurative disease caused by Staphylococcus aureus, which has important clinical and health significance. Monoclonal antibody against SEA used during the detection can effectively improve the sensitivity and specificity, but it is still not provided an effective anti-SEA monoclonal antibody in the domestic market.
To adapt to the clinical detection and basic research, we designed as following study: First, to improve the sensitivity and specificity of ELISA detection, we producing anti-SEA monoclonal antibody by fusing the spleen cells taking from BALB/c mice vaccinated with SEA four times with SP2/0 myeloma cells under 50%PEG. After screening with indirect ELISA assay and limited dilution cloning, three hybridoma cell strains were obtained, and named 3C1, 3G10 and 2A10 respectively, the results of identifications of which are followed. The three hybridoma cell strains with average number of chromosomes between 87～98, can steadily secrete antibodies after culturing consecutively four months and revitalization after six months cryopreservation. The antibodies are all IgG1 isotype with k light chain, and the cross-reactivity with staphylococcus enterotoxin B (SEB) or staphylococcus enterotoxin C1 (SEC1) have not shown using ELISA assay and Western blot analysis but bind to SEA specifically. The ELISA titers of monoclonal antibodies 3C1, 3G10, and 2A10 hybridoma cell strains which get from seroperitoneum are 1:819 200, 1:51 200, and 1:12 800 separately. The ability of relative affinity of the three hybridoma cell strains which recognize the same or similar speciesspecific epitope is 3C1>3G10>2A10.
An indirect competitive ELISA method based on the 3C1 monoclonal antibody detecting staphylococcus enterotoxin A in food was founded. The test conditions were optimized as followed: SEA (0.8μg/mL) were applied to 96 microtiter well plates(100/μL/well) and incubated at 37℃for 2h; the wells were blocked with 0.5% BSA in PBS at 37℃for 1 h. The titer of monoclonal antibody was 1:9 600, incubated at 37℃for 1 h with the ratio of the McAb:sample(70:30) while the reaction time of enzyme labeled antibody (1:6 000) and TMB was 30min and 10 min at 37℃. Under the qualification above, we got a standard curve, whose regression equation was y=-0.2902x＋0.8338 with the correlation coefficient r=0.9952. The detection limit of this method was 0.7311 ng/mL which can satisfy the requirement for detection SEA in foods, while coefficient variation (CV) of intra-assay and inter-assay were1.22%-13.47% and 9.31%～13.47% respectively suggested good reproducibility and accuracy. The indirect competive ELISA method based on 3C1 monoclonal antibody showed high specifity to SEA, but no cross-reaction with SEB or SEC1.We can detect SEA added in meat products at 0.8557ng/mL with the average recovery rate between the 60～120% and the CV of intra-and inter-assay〈20%.
According to the results above, indirect competitive ELISA method for detection SEA can be used for clinical test, this study is a groundwork for development of kit for detecting SEA.
本试验用SEA经多次免疫BALB／c小鼠后，取其脾细胞与SP2／0骨髓瘤细胞在50％PEG的作用下进行融合。用间接ELISA方法初步筛选出能分泌抗SEA单克隆抗体的杂交瘤细胞，用有限稀释法进行克隆化，直到克隆后的细胞孔达到100％阳性，即筛选到了只分泌一种抗体的杂交瘤细胞。本试验共筛选到三株杂交瘤细胞株，分别命名为3C1、3G10和2A10。对其进行鉴定，结果如下：在经4个月的连续传代培养和冻存6个月后复苏检测，其分泌抗体的能力差异不显著，表明其稳定性好；其染色体平均数目在87～98条之间；经亚类鉴定，三株杂交瘤细胞分泌的抗体类型均为IgG1亚类，轻链类型均为k链；ELISA交叉试验和Western-blot分析结果表明，三株抗金黄色葡萄球菌A型肠毒素的单克隆抗体与B型肠毒素、C1型肠毒素无任何交叉反应，且能特异性地与A型肠毒素结合；经抗原表位特异性分析，三株单克隆抗体的识别表位相同或相近；其相对亲和力的测定结果是3C1＞3G10＞2A10；抗体的效价水平为3C1＞3G10＞2A10，其中3C1株杂交瘤细胞的腹水效价达1∶819 200。以3C1杂交瘤细胞株的腹水型抗体为原料，建立了检测金黄色葡萄球菌A型肠毒素的间接竞争ELISA方法。通过对试验条件的系列优化，最终确定其反应条件如下：包被浓度为0.8μg／mL，包被条件为37℃2h；封闭条件为0.5％BSA封闭液37℃1h；抗体工作浓度为1∶9 600，样品和抗体的加样比为30∶70，竞争时间为37℃1h；酶标二抗浓度为1∶6000，反应时间为37℃30min；显色条件为37℃避光反应10min。在该反应条件下，获得一条标准曲线，其回归方程为y=-0.2902x+0.8338，相关系数r=0.9952，检测限为0.7311ng／mL，能满足目前对食品中肠毒素的检测要求。批内和批间变异系数分别为，1.22％～13.47％和9.31％～13.47％，具有较好的重复性和精确度；与B型和C1型肠毒素没有任何交叉反应，表明该方法的特异性强。用建立的检测A型肠毒素间接竞争ELISA方法对肉制品中添加的SEA进行检测。其定量限为0.8557ng／mL，平均回收率在60～120％之间，批内和批间变异系数均＜20％，说明本试验建立的检测金黄色葡萄球菌A型肠毒素间接竞争ELISA方法可用于临床检测，为金黄色葡萄球菌A型肠毒素检测试剂盒的研制奠定了基础，对预防葡萄球菌性食物中毒具有积极作用。
Staphylococcus aureus is a common pathogen that causes suppurative disease, and can produce staphylococcus enterotoxins, which are a leading reasons of food poisoning worldwide. Among all of the food poisoning caused by enterotoxins, staphylococcus enterotoxin type A (SEA) is the most important. Therefore, establishing a sensitive, rapid detection assay to SEA is necessary for food-safety, also contributing to early diagnosis of suppurative disease caused by Staphylococcus aureus, which has important clinical and health significance. Monoclonal antibody against SEA used during the detection can effectively improve the sensitivity and specificity, but it is still not provided an effective anti-SEA monoclonal antibody in the domestic market.
To adapt to the clinical detection and basic research, we designed as following study: First, to improve the sensitivity and specificity of ELISA detection, we producing anti-SEA monoclonal antibody by fusing the spleen cells taking from BALB/c mice vaccinated with SEA four times with SP2/0 myeloma cells under 50%PEG. After screening with indirect ELISA assay and limited dilution cloning, three hybridoma cell strains were obtained, and named 3C1, 3G10 and 2A10 respectively, the results of identifications of which are followed. The three hybridoma cell strains with average number of chromosomes between 87～98, can steadily secrete antibodies after culturing consecutively four months and revitalization after six months cryopreservation. The antibodies are all IgG1 isotype with k light chain, and the cross-reactivity with staphylococcus enterotoxin B (SEB) or staphylococcus enterotoxin C1 (SEC1) have not shown using ELISA assay and Western blot analysis but bind to SEA specifically. The ELISA titers of monoclonal antibodies 3C1, 3G10, and 2A10 hybridoma cell strains which get from seroperitoneum are 1:819 200, 1:51 200, and 1:12 800 separately. The ability of relative affinity of the three hybridoma cell strains which recognize the same or similar speciesspecific epitope is 3C1>3G10>2A10.
An indirect competitive ELISA method based on the 3C1 monoclonal antibody detecting staphylococcus enterotoxin A in food was founded. The test conditions were optimized as followed: SEA (0.8μg/mL) were applied to 96 microtiter well plates(100/μL/well) and incubated at 37℃for 2h; the wells were blocked with 0.5% BSA in PBS at 37℃for 1 h. The titer of monoclonal antibody was 1:9 600, incubated at 37℃for 1 h with the ratio of the McAb:sample(70:30) while the reaction time of enzyme labeled antibody (1:6 000) and TMB was 30min and 10 min at 37℃. Under the qualification above, we got a standard curve, whose regression equation was y=-0.2902x＋0.8338 with the correlation coefficient r=0.9952. The detection limit of this method was 0.7311 ng/mL which can satisfy the requirement for detection SEA in foods, while coefficient variation (CV) of intra-assay and inter-assay were1.22%-13.47% and 9.31%～13.47% respectively suggested good reproducibility and accuracy. The indirect competive ELISA method based on 3C1 monoclonal antibody showed high specifity to SEA, but no cross-reaction with SEB or SEC1.We can detect SEA added in meat products at 0.8557ng/mL with the average recovery rate between the 60～120% and the CV of intra-and inter-assay〈20%.
According to the results above, indirect competitive ELISA method for detection SEA can be used for clinical test, this study is a groundwork for development of kit for detecting SEA.
引文
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