日本血吸虫重组膜外蛋白rSj29免疫诊断的初步应用及代谢抗原单克隆抗体的制备
发布时间：2019-06-29 14:21
【摘要】： 血吸虫病在我国分布广泛,危害严重,至今仍是重要的公共卫生问题。快速,准确的早期诊断在血吸虫病的防治中起着重要作用。血吸虫病的诊断主要有病原学检查和免疫学检查,病原学诊断是确诊病人的主要方法,但费时,费力,对慢性感染和低度感染者容易漏检。因此寻找高度敏感、特异以及建立经济、可靠、能够考核疗效的免疫诊断方法依然是当前血吸虫病基础研究的重要内容。本实验室在前期已克隆表达了重组日本血吸虫四跨膜超家族蛋白(SjTsp2-A)和29 000膜外蛋白(Sj29),rSj29 ELISA在实验室用于血吸虫病人血清循环抗体的检测,获得较好的敏感性和特异性。我们重新纯化了rSj29蛋白和rSjTsp2蛋白,用30份混合阳性血清及30份混合阴性血清进行间接酶联免疫吸附实验(ELISA)的预实验,确定了最佳抗原包被浓度、二抗浓度。随后,从血吸虫病低度流行区芜湖南陵县采集394份粪便标本,男女随机,年龄5-80岁,改良加藤法三送九检,或集卵孵化法检测,同时采集其血清,进行间接血凝试验(粪便检查和间接血凝试验,由安徽省血吸虫病防治研究所完成),用rSj29间接ELISA法和AWA间接ELISA法检测血清相应抗体,每个样本均作复孔,采用单盲法检测,并与间接血凝试验和粪检方法进行了比较。结果显示阳性率:粪检,IHA,rSj29-ELISA,AWA-ELISA阳性率分别为4.82%(19/394),62.18%(245/394),68.27%(269/394)和89.85%(354/394)。其中血清学检测的阳性率均显著高于粪检(x2=1259.7,P0.01),IHA与AWA-ELISA符合率为63.20%,均为阳性者为227例,均为阴性者为22例; IHA与rSj29-ELISA符合率为80.71%,均为阳性者为219例,均为阴性者为99例。IHA与rSj29-ELISA的符合率较高。IHA与粪检结果比较,其敏感度,特异度,与粪检符合率分别为100%,39.73%,42.64%;rSj29-ELISA分别为94.74%,33.33%,36.04%;AWA-ELISA分别为100%,10.67%,14.97%。rSj29-ELISA与IHA敏感性(P=0.5)和特异性(x2=3.56,P0.05)差异无统计学意义。rSj29-ELISA与AWA-ELISA相比敏感性差异无统计学意义(P=0.5),特异性rSj29-ELISA高于AWA-ELISA(x2=55.98,P0.05)。表明rSj29间接ELISA法诊断日本血吸虫病与IHA相比具有相似的敏感性和特异性,且制备方法简单,易于标准化,可用于血吸虫病的免疫诊断初筛。 但ELISA和IHA法的特异性较低,抗体检测难以区分过去感染(包括经化疗治愈者)与现症感染,阳性率并非真实地反映现症病人。而循环抗原检测能更确切地反映疗效,更可靠地判断宿主体内有无活虫存在,提供准确的治疗依据及流行病学资料。随着单克隆抗体技术的出现和完善,现多用单克隆抗体技术进行循环抗原检测。 为此我们制备了抗日本血吸虫代谢抗原(Sj-MAg)的单克隆抗体。将尾蚴常规感染家兔,42天时处死家兔,门静脉灌注获得日本血吸虫成虫,通过体外培养制备了日本血吸虫成虫代谢抗原,鉴定该蛋白具有免疫反应性,能够识别日本血吸虫病人血清。蛋白免疫小鼠6周后,测定小鼠免疫效价达1:32 000以上。小鼠尾静脉注射纯蛋白以加强免疫,三天后取小鼠脾细胞与SP2/0骨髓瘤细胞进行杂交融合,HAT选择培养基筛选出杂交瘤细胞,间接ELISA法检测细胞培养上清,筛选阳性克隆,经三次亚克隆及扩大培养,获得了两株能够稳定分泌抗Sj-MAg的单克隆抗体的杂交瘤细胞株。采用Sigma抗体亚型检测试剂盒测定两株单克隆抗体的免疫球蛋白亚类均为IgG1。Western-Blotting实验确定了两株杂交瘤细胞株抗体能识别血吸虫代谢抗原。
[Abstract]:Schistosomiasis is a widespread and serious problem in China, and is still an important public health problem. Rapid and accurate early diagnosis plays an important role in the prevention and treatment of schistosomiasis. The diagnosis of schistosomiasis mainly includes the etiological examination and the immunological examination, the etiological diagnosis is the main method of the diagnosis of the patient, but it is time-consuming and labor-consuming, and it is easy to detect the chronic infection and the low-grade infection. Therefore, finding a highly sensitive, specific and economic, reliable and able to assess the immune diagnostic method of the curative effect is still an important part of the research on the basis of the current schistosomiasis. In this lab, the recombinant Schistosoma japonicum tetracross-membrane superfamily protein (SjTsp2-A) and the 29000 membrane outer protein (Sj29) were cloned and expressed in the earlier stage, and the rSj29 ELISA was used in the detection of the serum circulating antibody of the schistosomiasis human, and the better sensitivity and specificity were obtained. The rSj29 protein and rSjTsp2 protein were re-purified, and the optimal antigen coating concentration and the second anti-concentration were determined by using 30 mixed positive serum and 30 mixed negative serum for indirect enzyme-linked immunosorbent assay (ELISA). 394 stool samples were collected from Nanling County, Wuhu, a low-grade endemic area of schistosomiasis. The men and women were randomly selected, the age was 5-80 years old, the modified Kato method was sent to a nine-pass test, or the egg-collecting hatching method was used for the detection, and the serum was collected and the indirect hemagglutination test (faecal examination and indirect hemagglutination test) was carried out. The serum corresponding antibody was detected by indirect ELISA and AWA indirect ELISA, and each sample was made into a complex hole, and compared with the indirect hemagglutination test and the method of faecal examination. The positive rate of AWA-ELISA was 4.82% (19/394), 62.18% (245/394), 68.27% (269/394) and 89.85% (354/394) respectively. The positive rate of serologic test was significantly higher than that of the stool test (x2 = 1259.7, P0.01). The coincidence rate of IHA and AWA-ELISA was 63.20%. The coincidence rate of IHA and AWA-ELISA was 22 cases. The coincidence rate of IHA and rSj29-ELISA was 80.71%. The positive rate of IHA and rSj29-ELISA was 80.71%. The coincidence rate of IHA and rSj29-ELISA was higher. The sensitivity, specificity and the coincidence rate of IHA and fecal test were 100%, 39.73% and 42.64%, respectively; rSj29-ELISA was 94.74%, 33.33% and 36.04%, respectively; AWA-ELISA was 100%, 10.67%, 14.97%, rSj29-ELISA and IHA sensitivity (P = 0.5) and specificity (x2 = 3.56, P0.05) were not statistically significant. The sensitivity of rSj29-ELISA to AWA-ELISA was not significant (P = 0.5), and the specific rSj29-ELISA was higher than that of AWA-ELISA (x2 = 55.98, P0.05). The results show that the rSj29 indirect ELISA method has similar sensitivity and specificity as compared with the IHA, and the preparation method is simple, is easy to be standardized, and can be used for the immunodiagnosis of the schistosomiasis. However, the specificity of the ELISA and the IHA method is low, and the detection of the antibody is difficult to distinguish the past infection (including the treated by the chemotherapy) and the present disease, and the positive rate is not truly reflected. and the circulating antigen detection can more accurately reflect the curative effect, With the development and improvement of the monoclonal antibody technology, the multi-purpose monoclonal antibody technology is used for the circulation. Original test. For this purpose, we have prepared the anti-Japanese schistosome metabolic antigen (Sj-MAg). The monoclonal antibody of Schistosoma japonicum was sacrificed at 42 days, and the adult of Schistosoma japonicum was obtained by portal vein perfusion. The metabolic antigen of Schistosoma japonicum adult was prepared by in vitro culture. The immune titer of the mice was determined by 1: and after three days, the mouse spleen cells and the SP2/0 myeloma cells are hybridized and fused, the HAT selection medium is used for screening the hybridoma cell, the indirect ELISA method is used for detecting the culture supernatant of the cell culture, screening the positive clone, Two strains of monoclonal antibody capable of stably secreting anti-Sj-MAg were obtained by cloning and expanding culture. The immunoglobulin subclasses of two monoclonal antibodies were determined by using the Sigma-antibody subtype detection kit. Western-Blotting experiment confirmed the identification of two hybridoma cell lines.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R446.6;R392
[Abstract]:Schistosomiasis is a widespread and serious problem in China, and is still an important public health problem. Rapid and accurate early diagnosis plays an important role in the prevention and treatment of schistosomiasis. The diagnosis of schistosomiasis mainly includes the etiological examination and the immunological examination, the etiological diagnosis is the main method of the diagnosis of the patient, but it is time-consuming and labor-consuming, and it is easy to detect the chronic infection and the low-grade infection. Therefore, finding a highly sensitive, specific and economic, reliable and able to assess the immune diagnostic method of the curative effect is still an important part of the research on the basis of the current schistosomiasis. In this lab, the recombinant Schistosoma japonicum tetracross-membrane superfamily protein (SjTsp2-A) and the 29000 membrane outer protein (Sj29) were cloned and expressed in the earlier stage, and the rSj29 ELISA was used in the detection of the serum circulating antibody of the schistosomiasis human, and the better sensitivity and specificity were obtained. The rSj29 protein and rSjTsp2 protein were re-purified, and the optimal antigen coating concentration and the second anti-concentration were determined by using 30 mixed positive serum and 30 mixed negative serum for indirect enzyme-linked immunosorbent assay (ELISA). 394 stool samples were collected from Nanling County, Wuhu, a low-grade endemic area of schistosomiasis. The men and women were randomly selected, the age was 5-80 years old, the modified Kato method was sent to a nine-pass test, or the egg-collecting hatching method was used for the detection, and the serum was collected and the indirect hemagglutination test (faecal examination and indirect hemagglutination test) was carried out. The serum corresponding antibody was detected by indirect ELISA and AWA indirect ELISA, and each sample was made into a complex hole, and compared with the indirect hemagglutination test and the method of faecal examination. The positive rate of AWA-ELISA was 4.82% (19/394), 62.18% (245/394), 68.27% (269/394) and 89.85% (354/394) respectively. The positive rate of serologic test was significantly higher than that of the stool test (x2 = 1259.7, P0.01). The coincidence rate of IHA and AWA-ELISA was 63.20%. The coincidence rate of IHA and AWA-ELISA was 22 cases. The coincidence rate of IHA and rSj29-ELISA was 80.71%. The positive rate of IHA and rSj29-ELISA was 80.71%. The coincidence rate of IHA and rSj29-ELISA was higher. The sensitivity, specificity and the coincidence rate of IHA and fecal test were 100%, 39.73% and 42.64%, respectively; rSj29-ELISA was 94.74%, 33.33% and 36.04%, respectively; AWA-ELISA was 100%, 10.67%, 14.97%, rSj29-ELISA and IHA sensitivity (P = 0.5) and specificity (x2 = 3.56, P0.05) were not statistically significant. The sensitivity of rSj29-ELISA to AWA-ELISA was not significant (P = 0.5), and the specific rSj29-ELISA was higher than that of AWA-ELISA (x2 = 55.98, P0.05). The results show that the rSj29 indirect ELISA method has similar sensitivity and specificity as compared with the IHA, and the preparation method is simple, is easy to be standardized, and can be used for the immunodiagnosis of the schistosomiasis. However, the specificity of the ELISA and the IHA method is low, and the detection of the antibody is difficult to distinguish the past infection (including the treated by the chemotherapy) and the present disease, and the positive rate is not truly reflected. and the circulating antigen detection can more accurately reflect the curative effect, With the development and improvement of the monoclonal antibody technology, the multi-purpose monoclonal antibody technology is used for the circulation. Original test. For this purpose, we have prepared the anti-Japanese schistosome metabolic antigen (Sj-MAg). The monoclonal antibody of Schistosoma japonicum was sacrificed at 42 days, and the adult of Schistosoma japonicum was obtained by portal vein perfusion. The metabolic antigen of Schistosoma japonicum adult was prepared by in vitro culture. The immune titer of the mice was determined by 1: and after three days, the mouse spleen cells and the SP2/0 myeloma cells are hybridized and fused, the HAT selection medium is used for screening the hybridoma cell, the indirect ELISA method is used for detecting the culture supernatant of the cell culture, screening the positive clone, Two strains of monoclonal antibody capable of stably secreting anti-Sj-MAg were obtained by cloning and expanding culture. The immunoglobulin subclasses of two monoclonal antibodies were determined by using the Sigma-antibody subtype detection kit. Western-Blotting experiment confirmed the identification of two hybridoma cell lines.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R446.6;R392
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