人可溶性补体受体1型双抗体夹心ELISA法的建立及初步应用

摘要：目的：建立定量检测人血清可溶性补体受体1型（sCR1）双抗体ELISA夹心法。 方法：用鼠抗人CD35单克隆抗体(mAb)包被反应板,以兔抗人sCR1抗体（PcAb）为夹心抗体,辣根过氧化物酶（HRP）标记的山羊抗兔IgG为检测抗体,纯化后的人sCR1蛋白为标准品,建立定量检测人血清sCR1双抗体夹心ELISA法,并检测50例体检健康者和32例肝硬化患者血清样本中sCR1水平。 结果：建立的双抗体夹心ELISA法检测人血清sCR1的线性范围为15.6～250 ng/mL。低、高浓度批内变异系数（CV）分别为9.3％、7.1％;批间CV分别为11.2％和9.82％;回收率分别为89.5％和92.5％。50例健康人和32例肝硬化患者血清sCR1水平分别为（33.82±5.88） ng/mL和（152.99±9.51） ng/mL,两者比较有统计学差异（t=70.18, P＜0.001）。 结论：成功建立了检测人血清sCR1的双抗体夹心ELISA法,重复性和准确性较好。初步发现肝硬化患者血清sCR1水平显著升高。     

人血清哇巴因UPLC-MS/MS检测方法的建立和方法比对

目的建立超高效液相色谱串联质谱(UPLC-MS/MS)检测人血清哇巴因的方法。方法采用高特异性的UPLC-MS/MS,以氘标记的哇巴因-d3作为内标。样本采用固相萃取(SPE)前处理方法,以反相色谱柱负离子模式及电喷雾电离源(ESI)检测血清哇巴因水平。对建立的方法进行方法学(基质效应、回收率、准确度、批内精密度、批间精密度及稳定性)验证。采用建立的UPLC-MS/MS方法检测20名体检健康者及40例高血压患者血清哇巴因水平,并与酶联免疫吸附试验(ELISA)进行比较。结果 UPLC-MS/MS检测血清哇巴因的标准曲线范围为0.02～5.0 ng/mL,最低定量检测限(LLOQ)为0.02ng/mL。采用ABN固相萃取小柱进行样本前处理的基质效应较小,且回收率较高,达85%。LLOQ和低值(0.06 ng/mL)、中值(0.6 ng/mL)、高值(4 ng/mL)质控品的准确度分别为108.0%、89.2%、101.0%、103.0%。3个水平质控品的批内变异系数(CV)分别为2.87%、1.95%、0.56%,批间CV分别为5.98%、1.90%、0.75%。样本室温过夜放置16 h及样本前处理后室温放置自动进样器48 h的偏差均15%。采用UPLC-MS/MS检测哇巴因,正常对照者及高血压患者血清中均未检测到哇巴因。采用ELISA测定血清哇巴因,高血压患者为0.096 ng/mL,正常对照者为0.062 ng/mL。UPLC-MS/MS检测5个水平(0.02、0.05、0.10、0.20、0.50 ng/mL)的哇巴因标准品,其测定结果与对应的哇巴因标准品浓度呈正相关,且线性较好(r20.99),准确度较高;而ELISA检测5个水平哇巴因标准品的结果均很接近(0.024 9～0.029 6 ng/mL)。结论建立了检测人血清哇巴因的UPLC-MS/MS方法,未检测到正常人及高血压患者的血清哇巴因。UPLC-MS/MS与ELISA检测血清哇巴因的结果存在较大差异。     

血清层粘连蛋白荧光免疫层析检测方法的建立及性能评价

建立一种荧光定量层粘连蛋白(LN)检测方法,定量检测人血清LN水平。将鼠抗LN抗体用划线机喷到硝酸纤维素膜表面作为检测线(T),将链霉素亲和素用划线机喷到硝酸纤维素膜表面作为质控线(C);用生物素荧光微球标记鼠抗LN抗体作为缓冲液。利用双抗体夹心法原理定量检测LN水平,对方法的线性、精密度、空白限、回收率、相关性等性能指标进行验证,并使用市售化学发光LN试剂盒比对。荧光免疫分辨检测LN方法的回收率为86.03%~97.50%,空白限为4.22 ng/mL,线性范围为20~800 ng/mL,相关系数(r)为0.9989;批内精密度为6.51%,批间精密度为6.23%。与市售LN试剂平行检测100份血清标本,有良好的相关性(Y=1.0219X-1.1720,r=0.9871)。本研究建立了LN荧光免疫检测方法,各项性能指标均达到临床检验要求,操作便捷、结果准确,可用于临床血清LN水平的检测。     

不同ELISA系统检测人血清百日咳IgG抗体的比较研究

目的使用A、B、C 3组酶联免疫吸附试验(ELISA)系统进行百日咳疫苗临床血清检测,每组ELISA系统包括至少一种百日咳毒素(PT)、一种丝状血凝素(FHA)和一种黏附素(PRN)包被抗原,其中C组抗原包括2种FHA抗原,酶标二抗是相同的抗人IgG抗体。比较不同包被抗原对百日咳疫苗临床血清中3种IgG抗体检测结果的影响。方法收集164对无细胞百日咳、白喉和破伤风疫苗基础免疫前、免疫后临床血清,使用ELISA法检测和计算血清百日咳IgG抗体的转阳率和单位值。使用χ2检验比较转阳率差异,使用方差分析比较抗体检测结果。结果 A组抗原检测IgG抗体结果为PT 82.19IU/mL,FHA 81.66IU/mL,PRN39.34IU/mL;转阳率均为100.0%。B组抗原检测结果为PT 95.59IU/mL,FHA 57.39IU/mL,PRN 40.76IU/mL;转阳率PT抗体为99.4%,其余为100.0%。C组抗原检测结果为PT 60.74IU/mL,2种FHA分别为29.33IU/mL和53.32IU/mL,PRN 34.05IU/mL,转阳率为99.4%、95.1%、100.0%和98.8%。3组抗原检测的临床血清转阳率经χ2检验FHA差异有统计学意义(P0.05),PT和PRN差异无统计学意义(P0.05);抗体GMC经方差分析,所有组抗原检测3个抗体值之间差异均有统计学意义(P0.05)。结论本次人临床血清抗体转阳率和抗体值达到保护力水平,但是不同抗原检测系统之间差异均有统计学意义,应尽快建立百日咳抗血清IgG检测用抗原试剂国家参考品。     

定量检测人狂犬病毒中和抗体间接酶联免疫吸附测定方法的建立

目的 建立定量检测人狂犬病毒中和抗体间接酶联免疫吸附测定(ELISA)方法.方法 选择包被抗原和酶标二抗的最适浓度,用人狂犬病毒抗体标准品标化系列工作标准品,对临床收集的各类血清标本进行检测和分析.结果 间接ELISA定量检测中和抗体的最佳包被糖蛋白抗原浓度为10μg/L,酶标二抗的工作浓度为1:4 000,临床检验,间接ELISA定量方法灵敏度为100Vo.特异度为98.2%,总符合率99.0%;与快速荧光灶抑制实验呈(RFFIT)高度相关性(r=0.981).结论 本研究建立的定量检测狂犬病中和抗体方法,可以用于人狂犬病毒中和抗体的定量检测.     

新型肝损伤血清标志物Pep5定量检测方法的建立及临床应用

目的建立定量检测血清多肽Pep5的酶联免疫吸附方法(ELISA),用于判定肝损伤进程的研究。方法以待测血清样本稀释液包被酶联板,5%脱脂奶粉封闭,辣根过氧化物酶标记的抗Pep5单克隆抗体作为标记抗体,建立定量检测人血清多肽Pep5的ELISA,并用于临床检测690例肝损伤及健康血清样本。结果灵敏度为1.25ng/mL;线性范围(0～24ng/mL)内相关系数为0.998 7;与乙酰胆碱酯酶、层黏连蛋白、透明质酸、三型前胶原交叉反应率小于1.00%;添加回收率在99.09%～107.08%;批内、批间变异系数(CV)分别为5.62%～7.32%、6.21%～6.45%。与其他肝损伤指标乙酰胆碱酯酶试剂盒进行对比试验,表明两种检测方法有一定的相关性(r=0.68)。临床检测结果表明血清多肽Pep5水平随着肝损伤程度加重而升高,与正常血清差异有统计学意义(P0.01)。结论多肽Pep5可用于肝损伤进程定量血清学检测,检测方法简便、可靠,为后续深入研究多肽Pep5的功能及相关诊断试剂盒的研制和开发提供了科学依据。     

2007年浙江省嵊泗县健康人群麻疹、百日咳、白喉、破伤风监测分析

目的评价浙江省嵊泗县疫苗接种效果和人群免疫水平情况。方法随机抽取健康人群248名（包括个别外来流动人群）开展了人群麻疹、百日咳、白喉、破伤风抗体水平监测分析,用微量血球凝集抑制试验检测麻疹特异性IgG抗体,用试管凝集试验检测百日咳凝集抗体,用间接血凝试验检测白喉抗毒素和破伤风抗毒素。结果本次检测健康人群麻疹血清抗体阳性率为72.18%;百日咳血清抗体阳性率为80.74%,保护率为32.38%;白喉血清抗体阳性率为阳性率为54.25%;破伤风抗毒素达保护水平占72.18%。结论当前人群疫苗免疫保护水平尚有不足,可能与个别外来流动儿童未完成全程接种,个别疫苗接种程序、剂量改变以及海岛疫苗运输冷链条件差,个别批次疫苗质量是否可能下降等因素相关,值得进一步探讨。     

近红外荧光标记-免疫磁珠偶联法定量检测结核分枝杆菌ESAT-6蛋白

目的建立定量检测结核分枝杆菌早期分泌抗原靶-6(ESAT-6)的近红外荧光标记-免疫磁珠偶联法。方法以近红外荧光染料(Dylight 800)标记靶向ESAT-6的单克隆抗体,靶向ESAT-6的多克隆抗体包被在纳米磁珠表面。采用双抗体夹心法,磁性分离结合物和游离物,采用便携式高灵敏度低噪声激发式荧光检测仪检测磁性结合物的荧光强度,从而检测待检样品中ESAT-6水平。结果该方法的检测线性范围为2.4~750.0ng/mL,最低检测限为0.48ng/mL;10ng/mL水平加样回收率为96%,50ng/mL水平加样回收率为95%;批间变异系数(CV)为5.8%,批内CV为4.3%。该方法检测胸腔积液标本ESAT-6的特异度为80%,灵敏度为95%。结论该方法检测ESAT-6的线性范围广、灵敏度高、稳定性好。     

双抗体夹心ELISA测定人广泛型线粒体肌酸激酶方法的建立

目的建立测定人血清广泛型线粒体肌酸激酶（uMtCK）浓度的双抗体夹心酶联免疫吸附试验（ELISA）。方法应用原核表达的uMtCK重组蛋白免疫小鼠获得7株单克隆抗体,采用棋盘格滴定法筛选出最佳双抗体组合,其中抗体19F11进行辣根过氧化物酶（HRP）标记作为检测抗体,抗体3C1作为俘获抗体,建立检测人uMtCK双抗体夹心ELISA方法,并进行方法学评价。同时采用该法检测50名健康体检者和85例肿瘤标志物[甲胎蛋白（AFP）或癌胚抗原（CEA）]阳性患者的血清。结果通过筛查获得配对抗体,建立检测人uMtCK的双抗体夹心ELISA方法。该法的检测限为28．8ng／mL,批内变异系数（CV）为4．5％～7．7％,批间CV为8．2％～13．5％,平均回收率为97．1％。用该法检测50名健康体检者血清uMtCK浓度,结果均为阴性;85例肿瘤标志物阳性患者中有15例检出uMtCK,浓度为52．8～1442．2ng／mL。结论建立的双抗体夹心ELISA可以用于测定人血清uMtCK浓度。     

人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的评价与应用

目的对研制的人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的重复性、稳定性进行评价,并了解其实际应用效果。方法选取中、晚期肝硬化患者50例和健康体检者50例分别作为肝病组和正常对照组,以鼠抗人CD35单克隆抗体、兔抗人sCR1多克隆抗体和辣根过氧化物酶标记的山羊抗兔IgG为包被抗体、夹心抗体和检测抗体,以纯化后的重组人sCR1蛋白为标准品,建立人血清微量sCR1蛋白双抗体夹心ELISA试剂盒,进行试剂盒重复性和稳定性检验,并应用该试剂盒检测两组血清sCR1蛋白水平。结果双抗体夹心ELISA法检测人血清微量sCR1蛋白的线性范围是15.60~250.00ng/mL;血清sCR1蛋白水平对吸光度值的回归方程为Y=112.10 X2+18.21 X+1.694(r2=0.998);重复性检测中,高、低水平标准品检测值的批内相对标准偏差(RSD)分别为6.20%、7.40%,批间RSD分别为6.70%和7.90%;试剂盒稳定性检测中,RSD均不大于0.01;肝病组血清sCR1表达水平显著高于正常对照组(P0.01)。结论人血清微量sCR1蛋白双抗体夹心ELISA试剂盒线性范围广、重复性好、易于保存,适合于临床和科研检测工作。     

Estimating intra- and inter-assay variability in salivary cortisol

Investigators commonly assess intra- and inter-assay coefficients of variation (CVs) to estimate the precision of salivary cortisol enzyme immunoassay (EIA). However, little guidance is available as to which samples to use for CV assessment. The purposes of this methodological study were to compare differences in intra- and inter-assay CVs (a) among controls, standards, and/or unknown samples; and (b) between fresh and previously frozen saliva. A total of 174 duplicates (controls = 58, standards = 48, and unknowns = 68) were tested. The unknowns were from 34 students; all student saliva was assayed as both fresh and frozen samples. All samples were assayed in duplicate, using a commercial salivary cortisol EIA kit, by the same technician with the same equipment. A priori criteria for intra- and inter-assay CV, respectively, were ≤ 4% and ≤ 7%, and a was .05 for CV differences. Mean intra-assay CVs for controls, standards, unknowns, and combined samples were ≤ 2.5%, and mean inter-assay CVs were ≤ 2.8%. Mean intra-assay CVs were 2.2% for fresh saliva and 1.5% for frozen samples. Comparisons showed no significant differences in intra- or inter-assay CV among controls, standards, and/or unknown samples. Inter-assay CV was significantly different between fresh and previously frozen saliva (p = .043), with fresh saliva CV higher than frozen; the difference was not meaningful because all evaluations showed minimal measurement error. In conclusion, results indicate that estimation of precision can be achieved by testing of controls, standards, or unknowns and with either fresh or frozen saliva in this population.     

Quantitation of anti-tetanus and anti-diphtheria antibodies by enzymoimmunoassay: methodology and applications

We have developed enzymoimmunoassays (EIA) for the quantitation of antibodies (Ab) to tetanus and diphtheria toxoids (TT, DT) using Immulon I plates coated with the appropriate toxoid. A preparation of human tetanus immunoglobulin with a known concentration of anti-TT Ab was used as calibrator of the anti-TT antibody assay. The assay of anti-DT Ab is calibrated with a pool of human sera whose anti-DT Ab concentration was determined by quantitative immunoelectrophoresis, using a horse anti-DT with known Ab concentration as calibrator. A peroxidase-conjugated anti-human IgG was used in both assays. ABTS was used as substrate, and the reaction was stopped after 1 min incubation with citric acid and the OD measured at 414 nm on a Vmax reader. The assays have been applied to a variety of clinical situations. In patients suspected of having tetanus, the quantitation of antibodies has been helpful in establishing a diagnosis. In patients with a history of hypersensitivity to tetanus toxoid, verification of the levels of anti-TT antibody may prevent unnecessary and potentially harmful immunizations. The assays have also been used for the diagnostic evaluation of the humoral immune response to TT and DT, both in pediatric patients and in immunosuppressed patients. Several non-responders have been detected, and we have recently used the assay to monitor the effects of fish oil administration on the humoral immune response.(ABSTRACT TRUNCATED AT 250 WORDS)     

实时定量PCR检测胃癌组织miR-20a及初步应用

目的建立检测人胃癌组织中miR-20a的实时荧光定量PCR方法。方法建立miR-20a标准曲线,评价该法检测的线性范围及灵敏度;对标准质粒扩增产物进行熔解曲线分析和琼脂糖凝胶电泳检测以分析该法的特异性。取高、中、低3份不同浓度的标准质粒进行批内和日间变异系数(CV)的检测,分析该法的重复性。收集70例胃癌患者手术切除的癌组织和对应的癌旁组织,定量检测组织中miR-20a的表达水平。结果构建的实时荧光定量PCR法的灵敏度为101copies/μL,扩增的线性范围为101～109copies/μL;扩增产物的熔解曲线峰和电泳片段特异;3份不同浓度标准质粒定量检测的批内CV分别为6.33%、4.74%、5.89%,日间CV分别为9.45%、6.29%、7.48%。定量结果显示75.7%(53/70)胃癌组织中miR-20a的表达高于癌旁组织,差异有显著性(Z=-4.427,P<0.01)。结论建立的实时荧光定量PCR检测miR-20a的方法准确、快速、灵敏、特异,可用于胃癌组织中miR-20a的检测。     

陕西省铜川市健康人群百日咳、白喉、破伤风抗体水平监测结果分析

目的 了解陕西省铜川市健康人群百日咳、白喉、破伤风(百白破)抗体水平,为有效控制百白破提供参考依据。 方法 采用分层多级抽样的方法,在铜川市4区(县)随机抽取1岁、1~2岁、3~4岁、5~6岁、7~14岁、15~19岁、20岁健康人群368人,用酶联免疫吸附试验测定百日咳、白喉、破伤风IgG抗体。 结果 2011年铜川市健康人群百日咳抗体阳性率为38.04%,抗体几何平均浓度(GMC)为54.56 U/ml;白喉抗体阳性率94.02%,安全保护率60.87%,GMC为1.61 IU/ml;破伤风抗体保护率55.71%,GMC为2.70 IU/ml。 结论 铜川市健康人群百日咳IgG抗体阳性率低,7岁以上人群白喉及破伤风抗体保护率较低。预测铜川市近年不会发生白喉及新生儿破伤风疫情,但存在百日咳流行的隐患。故今后应通过加强百日咳的诊断及时规范管理病例,避免传播,还应按免疫程序及时加强免疫,并建议对大年龄组人群接种低白喉、破伤风联合疫苗含量的疫苗。     

2012年陕西省铜川市百白破疫苗免疫成功率监测分析

目的 评价陕西省铜川市百日咳-白喉-破伤风联合疫苗的免疫效果,为制定有效的免疫策略提供参考依据。 方法 用酶联免疫吸附试验方法检测适龄儿童全程免疫接种百白破疫苗前后百日咳、白喉、破伤风IgG抗体水平。 结果 全程免疫接种后,百日咳抗体阳转率为88.59%,抗体几何平均浓度(geometry mean concentration,GMC)增加了16倍;白喉抗体阳转率为100%,平均GMC增加了130倍;破伤风抗体阳转率为100%,平均GMC增加了57倍。 结论 百白破疫苗免疫效果良好,但百日咳菌苗免疫持久性较差。     

Development and validation of a choriogonadotropin beta-subunit radioimmunoassay for serum choriogonadotropin using commercially available components

A sensitive, specific, accurate and precise radioimmunoassay procedure developed for the beta-subunit of serum choriogonadotropin (hCGbeta) is described. 1. The assay employs an anti-hCGbeta (rabbit) serum generated against hCGbeta, highly purified intact choriogonadotropin (hCG) as standard, and [125I]hCG as the radioactive ligand. The antibody-bound hCG was separated from the free hormone by the addition of goat anti-rabbit gamma-globulin. 2. The detection limit of the assay was approximately 75 microIU hCG which corresponds to a serum concentration of approximately 0.75 mIU/mL. 3. Cross reactivity studies performed with human luteinizing hormone (hLH) and human thyroid-stimulating hormone (hTSH) indicated minimal interferences from these structurally similar glycoproteins. Parallel dose-response curves were demonstrated between dilutions of sera with elevated hCG concentrations and the standard reference preparation. A non-specific binding of less than 2.5% of the total [125I]hCG was routinely observed. 4. The analytical recovery of hCG added to human sera varied from 94 to 110%, with a mean recovery of 101%. 5. The inter-assay variation was determined by assaying (n=30) 3 different quality control pools. The following data were obtained: X1=4.6 +/- 0.5 mIU/mL (CV=10.9%); X2=8.1 +/- 0.9 mIU/mL (CV=11.1%); and X3=36.8 +/- 2.8 mIU/mL (CV=7.6%). 6. The clinical data collected from subjects with trophoblastic disease agreed with previously published studies. All of the reagents are available commercially.     

应用磺基水杨酸-硫酸钠比浊法在自动化分析仪上测定尿蛋白

目的对磺基水杨酸-硫酸钠(SS-S)比浊法测定尿蛋白的自动化分析进行评价。方法应用SS-S比浊法,在自动生化分析仪上建立测定参数,评价其方法的重复性、线性范围、回收率,并与氯化苄甲乙氧氨(BEC)法进行比较。结果批内和批间变异系数(CV)分别为0.87%~1.89%和0.76%~2.25%。线性范围为50.0~2 996.0 mg/L。回收率为97.3%~105.5%。与BEC法比较,YSS-S=1.001XBEC 16.3,相关系数(r)=0.978 8。结论SS-S比浊法测定尿蛋白具有较高的精密度和准确度,适合自动化分析。     

韶关市健康人群百日咳、白喉和破伤风抗体水平监测

目的　了解韶关市健康人群百日咳、白喉和破伤风免疫状况,及时为免疫策略提供科学依据。方法　随机抽查部分0～4 0岁健康人群进行了百日咳、白喉和破伤风抗体水平监测。结果　百日咳抗体几何平均滴度GeimetricMeanTiter(GMT)和抗体保护率分别为1∶374 2、67.74% ;白喉、破伤风抗毒素GMT分别为0.1699IU/ml、0.1804IU/ml,阳性率分别为88.94 %、91.83%。结论　对百日咳、白喉和破伤风已经形成了较好的免疫屏障,但人群百日咳抗体水平偏低,应大力推广应用吸附无细胞百日咳、白喉、破伤风联合疫苗.     

Tetanus immunization shortage in the United States

The purpose of this study is to determine the effect of the tetanus immunization shortage on EDs and the EPs understanding of the prioritization of persons needing tetanus immunization. A survey consisting of questions about knowledge of the tetanus shortage, prioritization of immunizations, incidence of tetanus infections, and understanding of CDC recommendations was mailed to a random sample 20% of the US ED medical directors. The results of the survey were input into the SPSS program (SPSS, version 10, Chicago, IL). The survey was returned by 618 of the 1,375 (44.9%) ED medical directors in the United States. Almost all (97.2%, 601 of 617) were notified about the tetanus shortage and 58.3% (360 of 617) reported a shortage. A total of 42.2% (199 of 472) gave tetanus toxoid (TT), instead of tetanus and diphtheria toxoids adult type (dT) when indicated. Only 11.6% of those surveyed (56 of 482) established a patient callback system. Routine vaccination was stopped in 37.5% of the reporting hospitals, most often for adults and children (57.5%, 69 of 120). Twelve hospitals (1.9%) reported they had an increase in tetanus. Although 87.5% of the respondents (539 of 616) stated they were familiar with the CDC's prioritization for tetanus immunization, only 1.8% (11 of 616) got the prioritization correct. Although EM directors uniformly know about and are experiencing the tetanus shortage, few correctly reported the tetanus immunization priority. Few EDs had a patient callback system.     

Sandwich enzyme-linked immunosorbent assay for plasma cholesteryl ester transfer protein concentration.

OBJECTIVES: Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesterol to apoB-containing lipoproteins. Its mass and activity are increased in several pro-atherogenic conditions. The objective of this study is to develop a cost- and time-effective sandwich ELISA for plasma CETP concentration. DESIGN AND METHODS: Monoclonal anti-CETP, TP20, was used as the capture antibody, while the other biotinylated monoclonal anti-CETP, TP2, was used for detection. The results were expressed in an arbitrary unit, ng biotin-TP2 bound per microl plasma. Plasma CETP concentrations, activities and their relationship were assessed in 35 IDDM children. RESULTS: The assay had an intra-assay CV of 8.75% and an inter-assay CV under 10%. Plasma CETP concentration of these subjects ranged from 0.36-1.89 ng biotin-TP2/microL. CETP concentration was significantly correlated with CETP activity (r = 0.51, p < 0.01). CONCLUSION: The sandwich ELISA we have developed carried sufficient sensitivity for assaying plasma CETP concentration in human.