【摘要】： 金葡菌腸毒素(Staphylococcal enterotoxins，SE)是一組由金黃色葡萄球菌分泌至胞外的水溶性蛋白，依據(jù)血清分型主要有腸毒素A、B、C、D、E等。其中腸毒素C又分為C1，C2，C3三個(gè)亞型。與普通抗原比，金葡菌腸毒素在很低濃度時(shí)就能活化的大量T細(xì)胞并產(chǎn)生強(qiáng)大免疫效應(yīng)，因此被認(rèn)為是超抗原。在腸毒素家族中，腸毒素A、B研究較多，腸毒素C則研究較少。 本研究的前期工作已從一種由金葡菌培養(yǎng)產(chǎn)物制得的生物制品-金葡素注射液的原液中提取了腸毒素，經(jīng)氨基酸序列測定和比較確定是腸毒素C2亞型，并通過藥效學(xué)試驗(yàn)證實(shí)了SEC2是金葡素注射液有效成分。由上述實(shí)驗(yàn)可認(rèn)為，SEC2含量是控制產(chǎn)品質(zhì)量的可信指標(biāo)。因此，建立一種簡便可靠的定量檢測SEC2的方法就十分必要。 本課題通過制備抗SEC2的特異性單克隆抗體，建立了檢測SEC2的雙抗體夾心ELISA法。采用雜交瘤單克隆技術(shù)，運(yùn)用已建立的間接ELISA法篩選，獲得四株分泌抗SEC2單克隆抗體(monoclonal antibody，McAb)的雜交瘤細(xì)胞株。四個(gè)雜交瘤細(xì)胞株分泌單克隆抗體的Ig類別均為IgG_1(k)，效價(jià)達(dá)1∶10~5—1∶10~7。采用辛酸-硫酸銨沉淀法純化各株雜交瘤細(xì)胞腹水，經(jīng)SDS-PAGE電泳分析，純化各株McAbs的純度均在80％以上。各株抗SEC2 McAbs能特異性識(shí)別SEC2，與牛血清白蛋白，人血清白蛋白及SEA、SEB、SEC1、SED型腸毒素?zé)o交叉反應(yīng)，僅McAb387與高劑量(＞1μg／ml)的SEC1有交叉反應(yīng)。采用非競爭法測得4株McAbs親和常數(shù)在1.1×10~9M~(-1)至2.46×10~(10)M~(-1)之間。應(yīng)用常規(guī)方法標(biāo)記純化McAbs，并經(jīng)阻斷抑制試驗(yàn)篩選McAbs識(shí)別位點(diǎn)，其中只有McAb 4F7和McAb 4D7之間識(shí)別抗原位點(diǎn)完全相關(guān)，其余McAbs之間識(shí)別抗原位點(diǎn)完全不相關(guān)。選擇二株識(shí)別不同抗原位點(diǎn)的抗SEC2McAbs，運(yùn)用于雙抗體夾心抗原的ELISA方法，，建立定量檢測SEC2的ELISA方法，靈敏度為0.5ng／L，批內(nèi)變異系數(shù)為3.71％至6.93％，批間平均數(shù)變異系數(shù)1.02％，回收率為97.8％至101％，回收率實(shí)驗(yàn)變異系數(shù)為2.2％至5.2％。應(yīng)用建立的單克隆抗體ELISA法測定了40批廠家送檢的金葡素注射液樣品中SEC2含量，同時(shí)與多克隆抗體ELISA法測定結(jié)果比較，結(jié)果顯示兩方法測定的SEC2含量無顯著差異，單克隆抗體法結(jié)果略低于多克隆抗體法。50批金葡素注射液的SEC2含量在5-16ng／ml之間，變異系數(shù)為16.8％。本研究成功的制備了抗SEC2 McAbs，建立了特異、準(zhǔn)確的定量檢測ELISA方法，并用于金葡素注射液制品的SEC2含量檢測，為超抗原研究和金葡素注射液質(zhì)量控制提供了有力的手段。 甘露(聚)糖結(jié)合凝集素(mannose-binding lectin or mannan-binding lectin；MBL)s是由肝臟分泌的具有膠原樣結(jié)構(gòu)的Ca~(2+)依賴的血漿C型凝集素，是先天免疫的重要組成部分。通過其糖基識(shí)別域MBL可識(shí)別并結(jié)合至很多微生物病原體的表面，隨后激活補(bǔ)體系統(tǒng)發(fā)揮中和和／或調(diào)理作用。作固有免疫成分和急性期蛋白，特別是在后天免疫系統(tǒng)成熟前階段和在感染后IgM出現(xiàn)之前，MBL都有重要意義。血循環(huán)中MBL含量水平主要由基因型決定，但許多非基因因素也對(duì)它有明顯影響。MBL不足或缺失與反復(fù)感染、反復(fù)流產(chǎn)、自身免疫病以及某些腫瘤等許多疾病相關(guān)。定量檢測MBL的試劑盒是進(jìn)行相關(guān)研究的必要條件，但目前MBL國內(nèi)沒有這種試劑盒，進(jìn)行MBL相關(guān)研究工作主要依賴進(jìn)口試劑盒，其價(jià)格昂貴，影響了國內(nèi)的研究發(fā)展。 本課題通過采用雜交瘤技術(shù)制備了兩株抗人MBL單克隆抗體(monoclonalantibody，McAb)并建立了MBL的定量ELISA檢測方法。研制了MBL檢測試劑盒，并初步應(yīng)用于檢測不同人群的MBL含量。以健康人血漿純化的MBL為抗原免疫小鼠，運(yùn)用已建立的間接ELISA法篩選，獲得兩株分泌抗人MBL單克隆抗體的雜交瘤細(xì)胞株。兩雜交瘤細(xì)胞株分泌單克隆抗體的Ig類別均為IgG_1(K)，效價(jià)達(dá)1∶10~6—1∶10~7，識(shí)別不同抗原位點(diǎn)。兩株McAbs能特異性識(shí)別MBL，與牛血清白蛋白，人血清白蛋白及由基因突變導(dǎo)致的MBL缺失的陰性血清無交叉反應(yīng)。采用辛酸-硫酸銨沉淀法和A蛋白親和層析法純化各株雜交瘤細(xì)胞腹水，經(jīng)SDS-PAGE電泳分析，純化各株McAbs的純度均在90％以上。應(yīng)用常規(guī)方法標(biāo)記經(jīng)純化的McAb-B3。運(yùn)用兩株McAb建立定量檢測MBL的ELISA方法，最佳檢測條件為8μg／ml濃度包被抗體B10，1∶500稀釋酶標(biāo)抗體B3。該方法特異、靈敏，有較好重復(fù)性。其準(zhǔn)確定量的檢測范圍為1～100ng／ml。批內(nèi)變異系數(shù)3.9％至5.2％，批間平均數(shù)變異系數(shù)為1.6％，均回收率為98.0％至105.3％，回收率實(shí)驗(yàn)變異系數(shù)為2.1％至4.3％。應(yīng)用建立的單克隆抗體ELISA法測定了52例健康人血清MBL含量，與丹麥試劑盒測定結(jié)果比較，兩方法測定的MBL含量有較好一致性。本研究的方法檢測范圍更寬。 用自建的MBL單抗ELISA夾心法對(duì)277例侗壯兩民族血清MBL含量水平進(jìn)行調(diào)查。結(jié)果經(jīng)統(tǒng)計(jì)學(xué)分析顯示，MBL含量侗族低于壯族(p＜0.01)，不同性別間無差異，不同年齡的MBL含量，在青春期明顯高于其它各時(shí)期(p＜0.01)，MBL缺失率在青春期明顯低于其他各時(shí)期，MBL缺失率與MBL含量存在負(fù)相關(guān)。對(duì)MBL低下的樣本進(jìn)行基因突變檢測，其中ExonⅠ54位密碼子突變51例，57位密碼子突變1例，沒有52位密碼子突變。所有突變的樣本其MBL含量均低于1μg／ml，MBL含量低下與ExonⅠ密碼子突變密切相關(guān)。另一調(diào)查檢測了204例兒童血清MBL水平，103例感染患兒(肺炎、腦炎、腹瀉、敗血癥)MBL水平明顯高于101例正常健康兒童，表明感染會(huì)導(dǎo)致MBL升高，MBL低水平可能與易發(fā)感染相關(guān)。
[Abstract]:Staphylococcal enterotoxins (SE) are a group of water-soluble proteins secreted by Staphylococcus aureus and extracellular. According to the serotype, SE is mainly composed of enterotoxins A, B, C, D and E. Enterotoxin C is divided into three subtypes, C1, C2, C3. Compared with common antigens, SE can activate a large number of T cells at very low concentrations and can activate them. Enterotoxins A and B have been studied more and enterotoxin C has been studied less in the enterotoxin family.
Enterotoxin was extracted from the original solution of Staphylococcus aureus injection, a biological product made from Staphylococcus aureus culture products. It was identified as enterotoxin C2 subtype by amino acid sequencing and comparison. The pharmacodynamic test confirmed that SEC2 was an effective component of Staphylococcus aureus injection. Therefore, it is necessary to establish a simple and reliable method for quantitative detection of SEC2.
A sandwich ELISA method for detecting SEC2 was established by preparing specific monoclonal antibodies against SEC2. Four hybridoma cell lines secreting monoclonal antibody (McAb) against SEC2 were obtained by using hybridoma monoclonal technique and indirect ELISA method. The antibodies were all IgG_1 (k) with titer of 1:10~5-1:10~7. The ascites of hybridoma cells were purified by octanoic acid-ammonium sulfate precipitation method. The purity of McAbs was above 80% by SDS-PAGE electrophoresis analysis. The anti-SEC_2 McAbs could specifically recognize SEC_2, bovine serum albumin, human serum albumin, SEA, SEB, SEC_1, SED type. Enterotoxin did not cross-react, but only McAb 387 did cross-react with SEC 1 of high dose (>1 ug/ml). The affinity constants of four McAbs strains were measured by non-competitive method between 1.1 (-1) and 2.46 (10) M (-1). McAbs was labeled and purified by routine method, and the McAb 4F7 and McAb 4D7 were screened by blocking inhibition test. Two strains of anti-SEC2 McAbs were selected to identify different antigen loci and applied to the ELISA method of double antibody sandwich antigen. An ELISA method for quantitative detection of SEC2 was established. The sensitivity was 0.5ng/L, the coefficient of variation in batch was 3.71% to 6.93%, and the mean variation between batches was 3.71% to 6.93%. The coefficients were 1.02%, the recoveries were 97.8% to 101%, and the experimental variation coefficients were 2.2% to 5.2%. The SEC2 content in 40 batches of Staphylococcus aureus injection samples was determined by the established monoclonal antibody ELISA method. The results showed that there was no significant difference in SEC2 content between the two methods. The results of monoclonal antibody assay were slightly lower than those of polyclonal antibody assay. The SEC2 content of 50 batches of Staphylococcus aureus injection ranged from 5 ng/ml to 16.8%, and the coefficient of variation was 16.8%. And Staphylococcal Enterotoxin C Injection quality control provides a powerful means.
Mannose-binding lectin or mannan-binding lectin (MBL) is a collagen-like Ca~ (2+)-dependent plasma lectin secreted by the liver and an important component of innate immunity. MBL is recognized and bound to the surface of many microbial pathogens through its glycosyl recognition domain and then activated. The complement system plays a neutralizing/regulating role. MBL plays an important role as an innate immune component and an acute phase protein, especially in the pre-mature stage of the acquired immune system and before the appearance of IgM after infection. Lack of MBL is associated with repeated infection, recurrent abortion, autoimmune diseases and some tumors. Quantitative detection of MBL kits is a necessary condition for related research. However, there is no such kit in MBL at present. The research on MBL mainly relies on imported kits, which is expensive and affects the development of domestic research.
Two monoclonal antibodies (McAb) against human MBL were prepared by hybridoma technique and a quantitative ELISA method was established for the detection of MBL. The MBL detection kit was developed and applied to detect the content of MBL in different populations. The mice were immunized with purified MBL from healthy human plasma using the established indirect ELIS method. Two hybridoma cell lines secreting monoclonal antibodies against human MBL were screened by A method. The two hybridoma cell lines secreted monoclonal antibodies of IgG_1 (K) with titer of 1:10~6-1:10~7, and recognized different antigen sites. The two McAbs could specifically recognize MBL, BSA, HSA and MB caused by gene mutation. The ascites of hybridoma cells were purified by octanoic acid-ammonium sulfate precipitation and A protein affinity chromatography. The purity of McAbs was above 90% by SDS-PAGE electrophoresis analysis. The purified McAb-B3 was labeled by routine method. The quantitative detection of MBL by two McAb strains was optimized by ELISA. The method is specific, sensitive and reproducible. The accurate quantitative detection range is 1-100ng/ml. The intra-assay coefficient of variation is 3.9% to 5.2%, the average inter-assay coefficient of variation is 1.6%, and the average recovery is 98.0% to 105.3%. The established monoclonal antibody ELISA method was used to determine serum MBL levels in 52 healthy subjects. The results of the two methods were in good agreement with those of the Danish kit.
The serum levels of MBL in 277 cases of Dong and Zhuang nationalities were investigated by self-made MBL monoclonal antibody ELISA sandwich method.Results Statistical analysis showed that the MBL content in Dong nationality was lower than that in Zhuang nationality(p<0.01),and there was no difference between the sexes.The MBL content in different ages was significantly higher than that in other periods during puberty(p<0.01). The MBL deletion rate was negatively correlated with the MBL content at other stages. The low MBL content was closely related to the Exon I codon mutation. Another survey detected serum MBL levels in 204 children, 103 infected children (pneumonia, encephalitis, diarrhea, sepsis) MBL levels were significantly higher than 101 healthy children, indicating that infection will lead to MBL increased, low MBL levels may be associated with susceptibility to infection.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李亞斐;細(xì)菌性超抗原的結(jié)構(gòu)及其免疫識(shí)別[J];國外醫(yī)學(xué).免疫學(xué)分冊;2002年05期

2 王艾麗;甘露聚糖結(jié)合凝集素的生物學(xué)功能及其臨床意義[J];醫(yī)學(xué)研究生學(xué)報(bào);2002年05期

3 王藍(lán)田;世界上第一個(gè)超抗原抗癌生物制劑——高聚金葡素的臨床效果[J];中國腫瘤臨床;1998年05期

4 王藍(lán)田;世界上第一個(gè)超抗原抗癌生物制劑——高聚金葡素的臨床效果[J];中國腫瘤臨床;1998年06期

5 蔣媛媛,鐘晉;高聚金葡素聯(lián)合化療治療惡性腫瘤的臨床觀察[J];現(xiàn)代診斷與治療;2004年04期

6 陳廷祚;高聚生研發(fā)歷程述評(píng)及其用于癌癥治療的理論基礎(chǔ)[J];微生物學(xué)免疫學(xué)進(jìn)展;2001年02期

7 陳月,陳政良,左大明,裘毅剛,張麗蕓;MBL對(duì)樹突狀細(xì)胞體外分化成熟的影響[J];細(xì)胞與分子免疫學(xué)雜志;2005年02期

8 賈天軍,李萍,孫黎,張艷芳,劉士先;MBL的提純及初步鑒定[J];張家口醫(yī)學(xué)院學(xué)報(bào);2003年01期

9 蘆玉蘭,余桂梅;高聚金葡素加鉑類治療復(fù)發(fā)卵巢癌合并腹水的臨床觀察[J];中國腫瘤臨床與康復(fù);2005年01期

10 王凡,黃強(qiáng),蘭青,周麗英,王愛東;高聚金葡素活化淋巴細(xì)胞對(duì)膠質(zhì)瘤的體外細(xì)胞毒作用[J];中國腫瘤臨床與康復(fù);2002年03期

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