小石榴
梅毒特异性IgG、IgM抗体分型检测方法
打印双面怎么设置的建立与临床应用
摘要
梅毒是一种常见的传播疾病，由梅毒螺旋体感染人体而发生，可累及全身各个器官且临床表现多样，具有极强的侵袭能力，严重危害到人类身体健康，是最为重要的性传播疾病之一[1]。
梅毒感染的病原体—苍白螺旋体（TP）很难在体外培养，因此，很难通过病原微生物培养进行诊断。梅毒诊断主要依据患者的临床表现和实验室检查结果[2-4]，包括以抗心磷脂抗体（反应素）为检测对象的非特异性类脂质抗原检测方法和以梅毒螺旋体抗体为检测对象的特异性检测方法两类。特异性检测结果只能反应梅毒特异性总抗体的存在情况。检测结果不能区分所检测抗体的类型，即不能判定是IgG还是IgM类型的抗体，也就不能判断患者是既往感染还是现症感染。梅毒特异性IgM抗
体的直接检测方法需要特殊的商品化试剂盒，操作步骤多，检测时间长，多为批量检测方法，无法在临床大规模应用。
本研究的第一部分：构建含梅毒螺旋体特异性抗原TP47基因片段的重组质粒，诱导表达TP47蛋白并纯化。将纯化后的TP47蛋白进行免疫印迹（weatern blot，WB）检测，对重组蛋白抗原性进行确认。结果显示：重组蛋白TP47能与梅毒阳性血清有特异性反应，与正常人梅毒阴性血清、自身免疫性疾病患者混合血清无反应。
本研究的第二部分：设计并制造一个能实现半自动化的亲和层析分离梅毒IgG和IgM抗体的装置，并验证装置的稳定性。制备亲和柱和脱盐柱，按照实验顺序分别装填入亲和装置中，优化亲和层析及脱盐条件。结果显示：亲和层析装置稳定性良好，无漏液、无管道污染、无非特异性吸附等问题；优化后亲和柱实验条件，即（1）样本上样液为500ul；（2）亲和柱的平衡液为20mM含5g/l的NaCL PH=7.5的Tris-HCL缓冲液4ml；（3）亲和柱的洗脱液为3M KSCN，洗脱体积为2ml。此时亲和填料可充分结合特异性梅毒抗体，并且平衡液4ml平衡后可去除非特异性IgG、IgM蛋白的残留；优化后脱盐柱实验条件，即（1）上样液为200ul的亲和柱洗脱液第2管；（2）脱盐柱的平衡液为20mM含5g/l 的NaCL PH=7.5的Tris-HCL缓冲液1ml；（3）脱盐柱的洗脱液为0.2M NaOH，洗脱体积为2ml。此时收集到的洗脱液中没有影响后续检测的
鹅饲料配方SCN-离子，便于后续样品检测；经脱盐柱后得到的样品收集液可用羊抗人IgG、IgM金标层析试纸条检测是否含有对应类型的抗体。
本研究的第三部分：用亲和层析装置检测临床230例梅毒总抗体阳性标本，并对其中40例标本用WB方法比对检测，对结果进行统计学分析。结果显示：40例标本与欧蒙公司WB方法检测的结果进行比较，两种检测方法结果无统计学差异（SPSS11.0软件、配对X2检验，p＞0.05）。
柳泉居士本课题通过通过自主研制的亲和层析装置，将梅毒螺旋体特异性抗原T1和TP58、TP56、TP47同时包被于亲和层析柱上，特异性地分离样本中IgG、IgM抗体，通过羊抗人IgG、IgM金标层析试纸条实现梅毒IgG、IgM抗体分型快速、准确检测，为临床对患者的梅毒现症感染提供诊断依据。同时用基因工程技术构建了一个特异性梅毒抗原TP47的重组表达蛋白并进行临床验证，为后续降低制造成本，实现一次性使用打下基础。
accord是什么意思
关键词：亲和层析；梅毒螺旋体；梅毒特异性IgG抗体；梅毒特异性IgM 抗体；特异性抗体分型检测
Establishment and Clinical Application for the Typing
Detection of Syphilis Specific IgG、IgM Antibody
曲阜师范大学研究生处ABSTRACT
解救的近义词Syphilis is a kind of common transmission dia,which is caud by Treponema pallidum infection.It can involve various organs and clinical manifestations,which has as a strong invasive ability,rious harm to human health.Syphilis is the most rious xually transmitted dias after AIDS[1].
Syphilis infection pathogens of Treponema pallidum(TP)is difficult to be cultured in vitro.So it is hard to diagno by pathogenic microorganism culture.Syphilis diagnosis is bad on clinical manifestations and laboratory findings,Including nonspecific lipid antigen(cardiolipin)detection anticardiolipin antibodies(reagin)specificity detection method and Treponema pallidum antibody as detection object[2-4].The results of laboratory tests can only detect syphilis specific antigen,the results can not distinguish the types of antibodies detected.We can not only determine that the type of the antibody is IgG or IgM,but also cannot judge with previous infection or current infection.Direct detection syphilis specific antibody IgM methods in the international all need special commercial kit,operation steps, long detection time,batch detection method,so we can not ues in large-scale clinical application.
The first part of this study:constructe recombinant plasmid of Treponema pallidum antigen TP47gene,induce the expression of TP47 protein and purify it.The purified TP47protein is tested by
WB to confirm recombinant protein.Result:The recombinant protein TP47was able to react
with the standard positive rum of syphilis,and did not react with normal human syphilis negative rum and Mixed ra from autoimmune dias patients.
散步作文The cond part of this study:Design and manufacture a device which can realize mi-automated affinity chromatography for parating syphilis antibody IgG and IgM,and verify the stability of the device.Prepare the affinity column and the desalting column,and respectively arranged them into the affinity device according to the experimental order,and optimize affinity and desalting conditions.Result:affinity chromatography device has good stability,no leakage,no pollution,no specific adsorption and so on; after optimized the experimental conditions of the affinity column:(1)the sample of affinity column was determined to be500ul;(2)The equilibrium solution of affinity column was20mM containing5g/l NaCL Tris buffer PH=7.5(4ml);(3)The affinity column was eluted with KSCN of3M2ml.At the same time,the affinity filler can be fully combined with the specific syphilis antibody,and the balance liquid can wash away the residue of non-specific protein after4ml;after optimized the experimental conditions of the desalting column:(1)10.the sample of the column was20
0ul affinity column eluent;(2)The equilibrium solution of the desalting column is20mM containing5g/l NaCL Tris buffer PH=7.5(1ml);(3)The elution of the desalting column was0.2M NaOH,2ml.At this time,the collected eluent had no influence on the subquent detection of SCN-ions,which was convenient for subquent sample detection;the samples collected from the desalination column can be detected by IgG and IgM gold labled strip.
The third part of this study:230cas of syphilis antibody positive samples were detected by affinity chromatography,and40cas of them was detected by WB.The results were statistically analyzed.Result:40cas detected by affinity device compared with the results by the WB method,the statistical results showed that two kinds of detection method results have no statistical difference(SPSS11.0software,paired X2test,p>0.05).
