日本血吸蟲(chóng)重組膜外蛋白rSj29免疫診斷的初步應(yīng)用及代謝抗原單克隆抗體的制備
發(fā)布時(shí)間：2019-06-29 14:21
【摘要】： 血吸蟲(chóng)病在我國(guó)分布廣泛,危害嚴(yán)重,至今仍是重要的公共衛(wèi)生問(wèn)題。快速,準(zhǔn)確的早期診斷在血吸蟲(chóng)病的防治中起著重要作用。血吸蟲(chóng)病的診斷主要有病原學(xué)檢查和免疫學(xué)檢查,病原學(xué)診斷是確診病人的主要方法,但費(fèi)時(shí),費(fèi)力,對(duì)慢性感染和低度感染者容易漏檢。因此尋找高度敏感、特異以及建立經(jīng)濟(jì)、可靠、能夠考核療效的免疫診斷方法依然是當(dāng)前血吸蟲(chóng)病基礎(chǔ)研究的重要內(nèi)容。本實(shí)驗(yàn)室在前期已克隆表達(dá)了重組日本血吸蟲(chóng)四跨膜超家族蛋白(SjTsp2-A)和29 000膜外蛋白(Sj29),rSj29 ELISA在實(shí)驗(yàn)室用于血吸蟲(chóng)病人血清循環(huán)抗體的檢測(cè),獲得較好的敏感性和特異性。我們重新純化了rSj29蛋白和rSjTsp2蛋白,用30份混合陽(yáng)性血清及30份混合陰性血清進(jìn)行間接酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)的預(yù)實(shí)驗(yàn),確定了最佳抗原包被濃度、二抗?jié)舛�。隨后,從血吸蟲(chóng)病低度流行區(qū)蕪湖南陵縣采集394份糞便標(biāo)本,男女隨機(jī),年齡5-80歲,改良加藤法三送九檢,或集卵孵化法檢測(cè),同時(shí)采集其血清,進(jìn)行間接血凝試驗(yàn)(糞便檢查和間接血凝試驗(yàn),由安徽省血吸蟲(chóng)病防治研究所完成),用rSj29間接ELISA法和AWA間接ELISA法檢測(cè)血清相應(yīng)抗體,每個(gè)樣本均作復(fù)孔,采用單盲法檢測(cè),并與間接血凝試驗(yàn)和糞檢方法進(jìn)行了比較。結(jié)果顯示陽(yáng)性率:糞檢,IHA,rSj29-ELISA,AWA-ELISA陽(yáng)性率分別為4.82%(19/394),62.18%(245/394),68.27%(269/394)和89.85%(354/394)。其中血清學(xué)檢測(cè)的陽(yáng)性率均顯著高于糞檢(x2=1259.7,P0.01),IHA與AWA-ELISA符合率為63.20%,均為陽(yáng)性者為227例,均為陰性者為22例; IHA與rSj29-ELISA符合率為80.71%,均為陽(yáng)性者為219例,均為陰性者為99例。IHA與rSj29-ELISA的符合率較高。IHA與糞檢結(jié)果比較,其敏感度,特異度,與糞檢符合率分別為100%,39.73%,42.64%;rSj29-ELISA分別為94.74%,33.33%,36.04%;AWA-ELISA分別為100%,10.67%,14.97%。rSj29-ELISA與IHA敏感性(P=0.5)和特異性(x2=3.56,P0.05)差異無(wú)統(tǒng)計(jì)學(xué)意義。rSj29-ELISA與AWA-ELISA相比敏感性差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.5),特異性rSj29-ELISA高于AWA-ELISA(x2=55.98,P0.05)。表明rSj29間接ELISA法診斷日本血吸蟲(chóng)病與IHA相比具有相似的敏感性和特異性,且制備方法簡(jiǎn)單,易于標(biāo)準(zhǔn)化,可用于血吸蟲(chóng)病的免疫診斷初篩。 但ELISA和IHA法的特異性較低,抗體檢測(cè)難以區(qū)分過(guò)去感染(包括經(jīng)化療治愈者)與現(xiàn)癥感染,陽(yáng)性率并非真實(shí)地反映現(xiàn)癥病人。而循環(huán)抗原檢測(cè)能更確切地反映療效,更可靠地判斷宿主體內(nèi)有無(wú)活蟲(chóng)存在,提供準(zhǔn)確的治療依據(jù)及流行病學(xué)資料。隨著單克隆抗體技術(shù)的出現(xiàn)和完善,現(xiàn)多用單克隆抗體技術(shù)進(jìn)行循環(huán)抗原檢測(cè)。 為此我們制備了抗日本血吸蟲(chóng)代謝抗原(Sj-MAg)的單克隆抗體。將尾蚴常規(guī)感染家兔,42天時(shí)處死家兔,門(mén)靜脈灌注獲得日本血吸蟲(chóng)成蟲(chóng),通過(guò)體外培養(yǎng)制備了日本血吸蟲(chóng)成蟲(chóng)代謝抗原,鑒定該蛋白具有免疫反應(yīng)性,能夠識(shí)別日本血吸蟲(chóng)病人血清。蛋白免疫小鼠6周后,測(cè)定小鼠免疫效價(jià)達(dá)1:32 000以上。小鼠尾靜脈注射純蛋白以加強(qiáng)免疫,三天后取小鼠脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行雜交融合,HAT選擇培養(yǎng)基篩選出雜交瘤細(xì)胞,間接ELISA法檢測(cè)細(xì)胞培養(yǎng)上清,篩選陽(yáng)性克隆,經(jīng)三次亞克隆及擴(kuò)大培養(yǎng),獲得了兩株能夠穩(wěn)定分泌抗Sj-MAg的單克隆抗體的雜交瘤細(xì)胞株。采用Sigma抗體亞型檢測(cè)試劑盒測(cè)定兩株單克隆抗體的免疫球蛋白亞類(lèi)均為IgG1。Western-Blotting實(shí)驗(yàn)確定了兩株雜交瘤細(xì)胞株抗體能識(shí)別血吸蟲(chóng)代謝抗原。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R446.6;R392
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