铁蛋白时间分辨荧光免疫分析法的建立

用夹心法建立铁蛋白(FER)时间分辨荧光免疫分析法(TRFIA),测定血清FER的含量。以FER单克隆抗体(McAb)806#包被板,双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合Eu^3 及标记FERMcAb 803#,发光增强系统为以β-二酮体为主的增强液。采用双对数函数数据处理程序,方法的批内和批间CV分别为2．2％和3．7％,平均回收率为118％,灵敏度为0．5μg／L,可测范围为0．5-1000μg／L。ED20、ED50和ED80分别为27．8μg／L、88．5μg／L和247μg／L。Eu^3 FER McAb于-30℃保存至少4个月,免疫反应性基本无损失,同批试剂连续分析4个月结果稳定。本法与PE公司的FER-TRFIA试剂盒所得样品的测定值的相关系数为0．98。本文建立的FER-TRFIA法,是一种适用于人群贫血普查和各种肝脏疾病及肿瘤的联合诊断的良好方法,分析范围、灵敏度和稳定性均较理想,值得推广应用。     

AFP时间分辨荧光免疫分析试剂盒的研制及其临床应用

目的:研制AFP时间分辨荧光免疫分析(TrFIA)试剂盒。方法:采用双抗体夹心法建立AFP-Tr-FIA试剂盒,对反应条件进行优化,并对试剂盒的各项指标进行评价。结果:抗体包被浓度确定为5μg/m l,铕标抗体最佳稀释比为1 50。试剂盒的线性范围为1ng/m l~1210ng/m l,灵敏度为0.41ng/m l,准确度高,批内和批间变异系数分别5.1%~8.8%,6.7%~11.9%。与CA199、CA125、CEA和白蛋白无交叉反应。破坏试验表明试剂在37℃可稳定7d。收集50例肝癌患者和370例健康人血清,用本试剂盒测得肝癌患者血清AFP浓度(557.3ng/m l±322.1ng/m l)显著高于健康人(6.5ng/m l±5.4ng/m l)(P<0.001)。370例正常人血清标本测试该试剂盒的正常参考范围为(0~12.0)ng/m l。本试剂盒检测结果与商用W allac AFP试剂盒检测结果相关系数为0.9988。结论:试剂盒各项指标达到规定的要求,可用于临床血清AFP检测。     

镧系元素时间分辨荧光免疫分析

一、概述 应用镧系元素的时间分辨荧光(TRF)分析技术,已在免疫检测中显示出它的优越性。在医学各学科中;如内分泌激素的检查、肿瘤标志物的检测,以及体内各种内或外源     

铁蛋白时间分辨荧光免疫层析法的建立及临床应用

建立铁蛋白(FER)时间分辨荧光免疫层析检测法(TRFIA-POCT)并进行临床应用。将FER抗体和兔IgG包被在硝酸纤维素膜(NC膜)上,以荧光微球标记抗FER单克隆抗体及兔IgG分别作为检测线和质控线制备荧光免疫层析试纸条。FER-TRFIA-POCT的灵敏度为0.5ng/ml;批内CV为3.2%~4.6%,批间CV为3.8%~5.2%;平均回收率为100.5%;热稳定性好;与电化学发光分析技术(ECLIA)比对,相关系数达0.8898。正常参考值范围男性为90~350ng/ml,女性为30~260ng/ml。本法建立的FER-TRFIA-POCT是一个快速高灵敏和可靠的检测。     

CEA时间分辨荧光免疫分析

以ＣＥＡ单克隆抗体Ｃ５０包被微孔板,异硫氰酸苄基二乙烯三胺四乙酸络合Ｅｕ＾３＋标记Ｃ１７单抗。采用平衡法建立ＣＥＡ时间分辨荧光免疫分析,数据采用Ｌｏｇ＝Ｌｏｇｉｔ函数数据自理程序处理。方法的批内和批间ＣＶ分别为２．９７％和１．６０％,平均回收率为１０１．９８％,灵敏度为０．２０μｇ／Ｌ,可测范围为２．３９～５０８．９μｇ／Ｌ,ＥＤ５０为６０．９１μｇ／Ｌ。本方法与ＡＦＰ和ＣＡ１２５和ＣＡ１５３无交     

基于纳米铕核铁蛋白时间分辨荧光免疫方法的建立

以人铁蛋白(ferritin,FER)为材料,制备纳米铕核微粒,并建立竞争型FER免疫检测方法。用稀盐酸解离FER的亚基后,纯化脱去游离铁,再通过酸碱中和方法重组蛋白外壳,同时将铕离子包裹于去铁蛋白内,制备铕核铁蛋白。经反应条件优化,确定pH 2.0为FER最适解离酸度。FER外壳重组时,铕试剂的添加量约为FER物质量的10 000倍时能获得最大捕获效率。以此建立了铕核-FER时间分辨荧光免疫检测方法,其灵敏度为0.576ng/mL,IC_(50)为13.15ng/mL,批间变异系数CV%<15%,非特异性结合率低。用制备成的铕核-FER构建的竞争法TRFIA与FER夹心法试剂盒对照,两者相关系数达0.966。通过上述方法获得免疫活性好、铕含量高的标记用纳米分子,简化了传统的铕标记方法,建立了构建纳米铕核铁蛋白的新方法。     

β2-MG时间分辨荧光免疫试剂盒的研制及质量考核

为建立灵敏度好、特异性高的TRFIA检测β2-MG,以羊抗鼠IgG固相抗体,Eu3 标记的β2-MG与标本β2-MG共同竞争限量的抗β2-MG鼠单克隆抗体(mAb),以Log-Logit制作标准曲线.本法检测灵敏度为0.01 mg/L,检测范围为0.1～8.0 mg/L;试剂及包被板在37℃放置7天后,监测标准曲线的ED20、ED50、ED80未见明显偏移;批内CV,批间CV分别为4.1%和7.8%,与美国PE试剂盒所测结果的相关系数为0.9364,说明两者具有良好的相关性.本法检测β2-MG灵敏度高,特异性好,试剂盒存放时间长且无放射性污染,是一种有应用前景的检测方法.     

乙肝病毒前S1抗原时间分辨荧光免疫分析试剂盒的研制

目的利用时间分辨荧光免疫分析（TRFIA）技术建立乙型肝炎病毒前S1抗原的检测试剂盒。方法应用双抗体夹心法建立乙肝preS1抗原TRFIA检测试剂盒,对试剂盒的各项性能指标进行评估。结果对280份血清样本进行检测并与国产酶联免疫法试剂盒对比,结果显示自制试剂盒的特异性更好：200份正常人血清样本检测结果表明,该试剂盒的cutoff值=阴性对照荧光值×4．5。用自制质控品检测分析内和分析间的精密度分别为3．4％～7．8％,6．9％-8.4％;检测HBsAg及HBeAg无交叉反应。结论试剂盒各项指标均达到临床检测要求,TRFIA法精密度和特异性有较大提高,可替代酶免法试剂盒。     

时间分辨荧光免疫分析法测定人血清α-FP

本文报告应用时间分辨荧光免疫分析技术测定人血清α-FP浓度。研究结果表明,将BAS引入该技术,使方法学稳定性和灵敏度有明显提高。本法批内CV为3.8～5.2％。批间CV为4.80～12.3％,最小检出值<1ng/ml,平均回收率为102.8％。健康成人测定参考值为5.56±5.38ng/ml(±SD)。聚苯乙烯微量滴定孔条经戊二醛处理后可提高包被抗体含量。     

A competitive dual-label time-resolved fluoroimmunoassay for the simultaneous detection of chlortetracycline and doxycycline in animal edible tissues

A dual-label time-resolved fluoroimmunoassay (TRFIA) was introduced for the simultaneous quantification of chlortetracycline (CTC) and doxycycline (DOX) in edible animal tissues. Europium- and samarium-labelled antibodies were used, because lanthanides have higher stabilities and narrower emission spectra than most fluorescent dyes. The limits of detection for simultaneous determination of CTC and DOX were 0.03 ng ml^?1 and 0.04 ng ml^?1, respectively. The average recoveries and the intra- and inter-assay coefficients of variation were 85.8–102.4%, 4.3–8.4% and 5.5–8.9%, respectively, and 85.3–101.6%, 4.5–9.2% and 5.3–9.6% for DOX, respectively. The proposed TRFIA for spiked samples was confirmed by high-performance liquid chromatography with a high correlation coefficient (R²) of 0.9933–0.9969. Therefore, the TRFIA may be an alternative sensitive and fast quantitative method for the high-throughput simultaneous screening of CTC or DOX in edible animal tissues.     

A comparison of time-resolved fluoroimmunoassay and ELISA in the detection of casein in foodstuffs

With the aim of lowering the detection limit for casein in foods, three competitive assays are described: direct time-resolved fluoroimmunoassay (TR-FIA), using europium-conjugated antibody, indirect TR-FIA, using biotinylated antibody with europium-conjugated streptavidin and ELISA, using a HRP-conjugated secondary antibody. Food samples (instant potato, flour mix, packet soup, spice-mix) were analysed. Standard curve sensitivities in direct and indirect TR-FIAs did not differ significantly (p=0.097), but both TR-FIAs were considerably less sensitive (both p<0.0001) than ELISA (LOQs 1.3, 1.5 and?<?1.0 mg kg^?1, respectively). The precision and working analyte range was similar in all three methods. Casein content measured in food products was comparable, using the three assays and rocket immunoelectrophoresis. The TR-FIA approach provided no improvement over the ELISA. All three assays allowed quantification of casein in foodstuffs in the order of 1–1.5 mg kg^?1, providing a basis for more rigorous validation and collaborative testing.     

甲状腺过氧化物酶抗体TRFIA技术的建立及临床应用

目的:建立甲状腺过氧化物酶抗体(TPOAb)时间分辨荧光免疫分析(TRFIA)竞争检测法。方法:用TPO抗原包板,用Eu3+标记羊抗鼠IgG做标记物,TRFIA检测人血清中的TPOAb。结果:TPOAb-TRFIA的灵敏度为1.0IU/ml;批内CV为3.2%～5.3%,批间CV为3.6%～5.6%;平均回收率为97.95%;热稳定性好;与电化学发光分析技术(ECLIA)比对,相关系数达0.9861;女性TPOAb测定值显著高于男性(P<0.01),且人群中TPOAb的阳性率女性也显著高于男性;TPOAb的正常值范围为≤32.6IU/ml。结论:本法建立的TPOAb-TRFIA是一个高灵敏和可靠的检测,有助于甲状腺疾病的临床诊断。     

总PSA时间分辨免疫荧光分析法的建立

利用时间分辨免疫荧光分析(TRFIA)技术建立人血清总PSA(前列腺特异性抗原)的快速全自动检测方法及其诊断试剂研制,该法采用双抗体夹心法建立tPSA-TRFIA,对该法和研制试剂的指标评价表明:方法的线性测量范围为0.50～250μg/L,灵敏度为0.06μg/L,批内批间的精密度分别为5.58%～9.71%,6.49%～12.12%。与CEA、CA12-5、CA15-3、CA19-9、AFP无交叉反应。353份血清标本用本试剂与国外其他试剂同时检测,其相关性达到94.4%,结果表明,试剂测定各项指标均达到临床检测要求,可替代国外同类产品。     

层粘连蛋白时间分辨免疫分析方法的建立

采用时间分辨荧光免疫分析(TRFIA)技术建立高灵敏的层粘连蛋白(LN)的快速的全自动检测方法.以羊抗鼠IgG抗体包被板,采用抗人LN单克隆抗体(McAb)、Eu3 标记人LN和以β-二酮体为主的增强液建立竞争LN-TRFIA,数据采用双对数函数处理程序处理.结果表明LN的标记率为11.5 Eu3 /LN,方法的批内和批间CV分别为3.9%和6.7%,平均回收率为91.2%,灵敏度为5μg/L,可测范围为5～1000μg/L,ED20、ED50和ED80分别为723.1μg/L、183.4μg/L和56.95μg/L.临床结果与RIA结果相符.本文建立的LN-TRFIA灵敏、稳定,可全自动操作,有很好的应用前景.     

Preparation of europium-streptavidin in a time-resolved fluoroimmunoassay for interleukin-3

The preparation and immunoassay performance of europium-labeled streptavidin is described. The Eu-streptavidin conjugate can serve as a general detection reagent in time-resolved fluoroimmunoassays (TRFIA). The usefulness of such a strategy has previously been demonstrated with the Eu3+ chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (Diamandis et al. 1988). In this report the conjugation of streptavidin was accomplished with the Eu3+ chelate of N1-(p-isothiocyanatobenzyl)diethylenetriamine-N1,N3,N3(+)-tetraace tic acid, using a 50 M excess of the label. The conjugation ratio of Eu3+/streptavidin was 16. The use of the Eu-streptavidin reagent in a two-site immunometric assay to measure human recombinant interleukin-3 in human plasma, showed that the useful range of the assay was 20-25,000 pg/ml.     

Comparison of the determination of chloramphenicol residues in aquaculture tissues by time-resolved fluoroimmunoassay and with liquid chromatography and tandem mass spectrometry

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of chloramphenicol (CAP) residues in aquaculture tissues based on monoclonal antibodies. The limits of detection (LOD) were determined to be 0.04 ng g^?1 and the limits of quantification (LOQ) were less than 0.15 ng g^?1. The intra-assay variations were below 10% and the interassay variations ranged between 9.7 and 13.3%. The mean recoveries established at six concentration levels varied from 83.7–109.6% and the coefficient of variation was from 8.3–11.5%. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC-MS-MS). This proposed technique could be applied to routine residue analysis.     

HBeAg时间分辨荧光免疫分析法的建立

采用平衡饱和法建立了乙型肝炎病毒e抗原(HBeAg)时间分辨荧光免疫分析法.以针对HBeAg的单克隆抗体G8包被板,双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合Eu3+及标记C4单抗,发光增强系统为以β-二酮体为主的增强液.数据采用Log-Logit法函数和四参数Logitc函数数据处理程序处理.结果表明方法的批内和批间CV分别为2.39%和5.28%,平均回收率为97.62%,灵敏度为0.58NCU/mL,可测范围为12.01-529.84NCU/mL,ED20、ED50和ED80分别为6.36NCU/mL、26.85NCU/mL和136.7NCU/mL.本方法与HBsAg有13.1%的交叉反应.Eu3+标记抗体-30℃保存6个月免疫反应性基本无损失,同批试剂连续5个月应用分析结果稳定.HBeAg时间分辨荧光免疫分析的质量参数优于EIA和IRMA.     

Comparison of the determination of chloramphenicol residues in aquaculture tissues by time-resolved fluoroimmunoassay and with liquid chromatography and tandem mass spectrometry

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of chloramphenicol (CAP) residues in aquaculture tissues based on monoclonal antibodies. The limits of detection (LOD) were determined to be 0.04 ng g^-1 and the limits of quantification (LOQ) were less than 0.15 ng g^-1. The intra-assay variations were below 10% and the interassay variations ranged between 9.7 and 13.3%. The mean recoveries established at six concentration levels varied from 83.7-109.6% and the coefficient of variation was from 8.3-11.5%. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC-MS-MS). This proposed technique could be applied to routine residue analysis.