Establishment of Double-antigen Sandwich Time-resolved Fluorescence Immunoassay for Detection of Pest des Petits Ruminants Virus

[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.

The Development of A Fluorescence Polarization Immunoassay for Aflatoxin Detection

A fluorescence polarization immunoassay （FPIA） was developed for the analysis ofaflatoxins （AFs） using an anti-aflatoxin B1 （AFB1） monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 rain. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.

3,6-Bis-β-Dicarbonylsubstituted Carbazoles Bearing N-Spacers and Their Eu(III) Complexes as Immunofluorescent Labelling Agents

New reagents for immunofluorescence analysis of carbazole series containing fluorinated β-dicarbonyl fragments and carboxylic substituent groups separated by spacers of different lengths from the light-gathering carbazole scaffold have been developed. The markers in complex with Eu3+ ions possess stability in the aqueous phase, intense and prolonged luminescence (τ 550 - 570 μs) with characteristic emission maxima in the region of 615 nm and excitation wavelengths in the region of 380 - 390 nm, which distinguishes them from most of the analogs used. In the study of marker conjugation with streptavidin, a reagent containing 4 - 5 europium labeling complexes based on spacer-containing carbazole tetraketone was obtained. The marker-doped silicate nanoparticles exhibit intense and long-lived luminescence in the characteristic region.

Production of Polyclonal Antibody of Morphine and Determination of Morphine in Urine by Capillary Electrophoresis Immunoassay with Laser-induced Fluorescence Detection

N-Conjugated antigen was synthesized and polyclonal antibody with high specificity was obtained from immunizing animals. With this polyclonal antibody, a rapid and efficient CEIA-LIF method was developed to determine the free morphine in urine of abusers. The detection limit was calculated to be 40 ng/mL. Simulated urine samples were analyzed with good recoveries, which showed the feasibility of its application in specific morphine determination in urine of morphine abusers.

Antibody Production for a Rapid Fluorescence Polarization Immunoassay of Estrone

Estrone has been identified as a potential endocrine-disrupting chemical （EDC）[1]. Estrone is usually quantified by gas chromatography-mass spectrometry （GC-MS）, GC-MS/MS, high performance liquid chromatography （HPLC）, HPLC- MS, and HPLC-MS/MS, etc.[2-3]. Meanwhile, several immunoassays based on radioimmunoassay, enzyme linked immunosorbent assay （ELISA） or chemiluminescence immunoassay （CLIA） for determination of estrone in real samples have been developed[2＇4]. Although these methods are sensitive, they need multistage separation and are thus time-consuming and laborious. A very promising way for the simplification of immunoassays for routine applications is a shift from heterogeneous methods （with separation） to homogeneous assays （without separation）[5]. Fluorescence polarization immunoassay （FPIA） is one of the homogeneous techniques that meets the requirements of a simple, reliable, fast, and cost-effective analysis[6]. Therefore, the present study is focused on the development of FPIA in order to analyze estrone based on antibody production.

A QUENCHING FLUORESCENCE IMMUNOASSAY METHOD FOR DETERMINATION OF TRACE ALBUMIN USING UNLABELED TERBIUM CHELATE

A fluoroimmunoassay method using unlabeled Terbium chelate is described.The principle is similar to that of fluoroimmunoassay method using lanthanide chelate as labels.The procedure is simpte because labeling process is unnecessary.The recovery of HSA and albumin in urine is 107% and 95% respectively.The standard deviation is tess than 10%.

动物源食品中孕酮激素残留的时间分辨荧光免疫分析法的建立

该研究以孕酮为研究对象,设计并制备了新抗原,通过动物免疫得到特异性兔抗体,基于所得抗原和抗体,进一步优化了抗原抗体浓度、反应缓冲液种类及其pH值等关键参数,最终在国内首次建立了用于动物性食品中性激素孕酮残留快速检测时间分辨荧光免疫分析法(TRFIA)。该研究确定最优实验条件为:抗原0.30μg/mL、铕标抗体0.40μg/mL、pH值7.2的TBST工作缓冲液,并建立标准曲线,方法的线性范围为0.46~6.94μg/L,IC_(50)达1.79μg/L,与睾酮交叉反应率为1.25%,与雌二醇、雌三醇等激素无交叉反应。实际样品的平均添加回收率为82.0%~113.0%,批内批间变异系数<5%,与高效液相色谱法(HPLC)检测结果的相关性良好(r2=0.988)。结果表明该研究所建立的TRFIA检测方法准确可靠、特异性好、灵敏度高,完全可用于动物源食品中孕酮残留的快速检测。

时间分辨荧光免疫分析仪的校准方法研究

研究一种适用于时间分辨荧光原理的免疫分析仪校准方法。经过对时间分辨荧光免疫分析仪荧光特性、孵育舱温度、分析项目浓度示值误差,重复性等性能指标的实验和研究,建立该分析仪的校准方法。起草该分析仪的校准规范,提出了仪器的校准项目与技术指标,确定了校准用的仪器设备,设计了完备的校准方案,使时间分辨荧光免疫分析仪的量值准确、可靠、可溯源,为统一相关量值发挥重要的作用。

用于诺氟沙星现场灵敏检测的便携式智能手机赋能荧光传感技术

通过融合激光诱导荧光原理和光纤传感原理,集成全光纤光学系统、微型化流动进样系统、高灵敏微型光电探测系统和基于智能手机的APP软件,研制了用于水中抗生素现场快速灵敏检测的便携式智能手机赋能荧光生物传感器.以喹诺酮类抗生素诺氟沙星(NOR)为例,在优化条件下,该荧光生物传感器对NOR的检测限可达0.35μg/L,线性检测范围为1.6~20.6µg/L.所建立的NOR检测方法也被用于各种水样的NOR加标回收检测,其回收率为85%~120%,检测时间少于15min,表明其可以用于实际样品中NOR的现场灵敏快速检测.结合物联网等新兴技术,可根据设定的程序将检测结果直接上传环境监测监管中心,为实现水环境安全的监测预警和应急监测提供重要技术支持.

Developing a fluorescence substrate for HRP-based diagnostic assays with superiorities over the commercial ADHP

The combination of horseradish peroxidase(HRP)and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays.10-Acetyl-3,7-dihydroxyphenoxazine(ADHP or Amplex red)is the most sensitive fluorogenic substrate known for HRP in current market,however,it suffers from some drawbacks,such as non-specific reactivity to carboxylesterase and limited fluorescence stability.In the present study,a novel HRP substrate10-cyclopropylcarbonyl-dichloro-dihydroxyphenoxazine(AR-2),has been prepared,which exhibited improved sensitivity than ADHP in sensing HRP.Moreover,the fluorescence of AR-2/HRP demonstrated improved tolerance to physiological relevant p H fluctuation as compared to ADHP/HRP.Successful detection of uric acid/urate oxidase reaction indicated excellent application prospect of AR-2/HRP for monitoring H_(2)O_(2)-generating biochemical reactions.More interestingly,an enzyme-linked immunosorbent assay(ELISA)using AR-2 as the fluorescence reporter has been successfully used in detecting IgG against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)from human serum samples.Overall,AR-2 exhibits improved performances over the commercial ADHP,which will be an ideal alternative to ADHP in HRP-based fluorescence biochemical analysis and immunoassays.

我国免疫分析仪发展现状与趋势研究

免疫分析技术作为疾病分析的重要手段,一直是生命科学领域研究的重点。近些年随着科学技术的不断发展,免疫诊断已逐渐成为体外诊断行业最大的细分领域。免疫分析仪作为免疫诊断的定量分析仪器,其发展规模、技术水平影响着免疫诊断行业高质量快速发展的程度。本文概述了免疫分析仪行业的发展现状,分析了各细分领域的注册情况以及竞争格局,总结了免疫分析仪行业存在的问题,并对行业的未来发展趋势进行了展望。

新型均相免疫分析技术——荧光淬体免疫探针分析

生化分析在疾病诊断、环境污染源鉴定、食品安全等多个领域具有重要意义。基于抗原抗体反应的免疫检测技术具有高特异性与多样性等特点,其操作比基于大型设备的色谱法简单,检测对象选择性更好,广泛应用于样品检测。荧光淬体免疫探针(Q-body)是一种通过荧光染料标记抗体片段构建成的新型荧光免疫检测探针,抗体内部色氨酸导致荧光基团发生电子转移而淬灭,当与抗原特异性结合时,其荧光强度恢复并呈浓度依赖性。因此,可通过检测Q-body的荧光强度变化检测抗原浓度。Q-body免疫检测技术因操作简便及灵敏度高等特点,被广泛应用于环境污染物质及疾病标志物的快速检测,对环境检测及疾病诊断具有重要意义。本文从Q-body的原理、构建方法、检测技术的开发及国内外研究进展进行综合论述,并对该技术的未来应用做出展望。

免疫测定法检测农药残留的研究进展

农药除被作物靶向部位吸收外,剩余部分的农药富集、渗透到土壤或空气中。因此,利用可靠、准确且有效的检测技术来监控农药残留尤为重要。该文综述检测不同种类农药残留的免疫分析方法,包括酶联免疫吸附分析法、荧光免疫分析法、化学发光免疫分析法、免疫层析法和放射免疫分析法,对上述技术的基本原理和用途进行讨论和对比,并阐述不同种类农药免疫测定技术的发展潜力与趋势。

S100B时间分辨荧光免疫检测试剂盒的制备及性能评价

目的:制备一种快捷、准确、定量检测血清中S100B蛋白浓度的时间分辨荧光免疫检测试剂盒,并对其进行性能评价。方法:使用时间分辨荧光微球标记的抗S100B多克隆抗体及兔IgG抗体、标记垫、样品垫、S100B硝酸纤维素膜板、吸水纸制备试纸条,组装卡壳得到S100B时间分辨荧光免疫层析试剂盒。通过标准曲线、准确度、最低检测限、线性区间、特异度、重复性、稳定性等检测对该试剂盒进行性能评估。采用该试剂盒对某地区199例健康者血清及血浆样本的参考区间进行研究,并采用同步盲法测试该试剂盒与罗氏Elecsys S100检测试剂盒的临床应用性能,评估2种试剂盒对于142例样本检测结果的一致性。结果:该试剂盒所得标准曲线方程为y=(1.13302+1.75224)/[1+(x/1.08220)×(-0.60352)]-1.75224,R2=0.99908,线性范围为[0.05,30]ng/mL。该试剂盒可满足准确度相对偏差在±15%内、最低检测限≤0.05 ng/mL、特异度相对偏差在±15%内、批内差和批间差的变异系数均<15%的产品设计要求。稳定性检测结果标明该试剂盒可在2~30℃条件下有效保存12个月。同时,通过该试剂盒测得血清及血浆样本参考区间均<0.3 ng/mL。临床验证结果表明该试剂盒与罗氏Elecsys S100检测试剂盒的测定结果具有高度一致性(Kappa=0.9061>0.80),符合标准。结论:该试剂盒可快捷、准确、定量检测血清中S100B蛋白浓度,为神经类疾病、自身免疫病性疾病、脑血管疾病等的诊断与治疗提供了参考。

基于荧光免疫法的地表水中2,4-D原位快速检测

采用荧光免疫法原位测定地表水中2,4-二氯苯氧乙酸(2,4-D),方法在7.2μg/L~211.7μg/L范围内线性良好,最低检出限为2.6μg/L。对空白水、长江水和湖泊水样品做2,4-D的4个质量浓度水平的加标回收试验,7次测定结果的RSD为3.9%~5.8%,加标回收率为85.9%~106%。用该方法与国标法同时测定黄石市内某长江水源地和某湖泊水中的2,4-D,结果无明显差异。

三种方法动态监测儿童肺炎支原体感染的比较及药物敏感性分析

目的探究三种方法动态监测儿童肺炎支原体(MP)感染的比较及药物敏感性分析。方法选取本院2020年6月到2023年10月期间收治的临床诊断为下呼吸道感染的患儿300例作为此次研究对象。采用MP培养及药敏试验、化学发光免疫法(CLIA)以及MP荧光定量PCR法对MP感染阳性率进行检测。结果300例下呼吸道感染患儿经过MP-IgM抗体检测,确诊为MP的患儿有196例(65.33%)。MP培养阳性检出率为58.67%(115/196),CLIA法阳性检出率79.59%(156/196),MP荧光定量PCR阳性检出率65.31%(128/196)。CLIA法检测MP感染阳性率较MP培养和MP荧光定量PCR高,差异有统计学意义(χ^(2)=15.676,P<0.05)。MP培养、CLIA法和MP荧光定量PCR与金标准的一致性尚可。4~6岁组患儿阳性检出率最高,为69.38%,高于其他年龄组。而CLIA法检测MP阳性最为灵敏,在4~6岁组患儿中阳性检出率高达54.08%。病程<7天组阳性检出率71.34%高于≥7天组58.74%。其中CLIA法检测最为灵敏,在<7天组和≥7天组中检出率分别为83.93%、73.81%,三种方法比较有差异(P<0.05)。MP对阿奇霉素、左氧氟沙星、加替沙星、司帕沙星敏感度较高,对红霉素、罗红霉素、乙酰螺旋霉素耐药度较高。结论MP培养、CLIA法和MP荧光定量PCR检测儿童MP均有较好的应用效果,而CLIA法检测更具优势,临床治疗时应对MP敏感度较高的常用抗菌药物优中选优。

化学发光法与乙肝病毒DNA荧光定量在乙肝检测中的应用价值

目的:明确化学发光法(CLIA)、乙肝病毒DNA荧光定量技术在乙肝检测中的价值。方法:选取285例乙肝患者和280例肝硬化患者为检测对象,分别对样本进行CLIA、乙肝病毒DNA荧光定量分析,统计相关检测数据。结果:乙肝组HBsAg、HBsAb、HBeAg、HBeAb、HBcAb水平均明显低于肝硬化组(P<0.05),HBV-DNA水平明显高于肝硬化组,随着HBV-DNA水平上升,乙肝患者的肝功能指标均有不同程度提高(P<0.05)。经Pearson相关性分析,HBV-DNA和HBsAg、HBsAb、HBeAg、HBeAb、HBcAb均为负相关(t=-0.876、-0.956、-0.974、-0.922、-0.981)。结论:CILA、乙肝病毒DNA荧光定量检测技术在乙肝检测中都有一定价值,两者之间为负相关,可为乙肝诊断、预后评估提供参考信息。

全自动酶标免疫分析仪的技术原理与故障案例分析

分析全自动酶标免疫分析仪的技术原理及典型故障案例,以提升全自动酶标免疫分析仪的使用效果。通过微粒子酶免疫分析(MEIA)技术完成酶免试验,以主定标、标准定标、定性定标及定标液修正为全自动酶标免疫分析仪的定标方式,提升酶免试验分析精度。通过分析全自动酶标免疫分析仪使用中遇到的洗板机、开机初始化报警、启动软盘、加样臂和滤光器等典型故障案例,提出故障解决应对措施,为全自动酶标免疫分析仪后期维修与管理提供参考。

干式免疫荧光定量分析法检测降钙素原与血培养结果的关系研究

目的分析与讨论干式免疫荧光定量分析法检测血培养、降钙素原(PCT)的关系和价值。方法筛选2021年1月至2023年2月64例血流感染(BSI)患者为研究对象,均检验PCT水平及血培养,对血培养阴性和阳性结果患者PCT水平对比,同时分析阳性患者病原菌分布情况,对比不同病原菌感染患者PCT指标间的差异。结果64例患者经血培养检验,12例患者结果呈阳性,占18.75%,52例阴性,占81.25%;血培养结果为阳性患者PCT水平高于阴性者,差异有统计学意义(P<0.05),12例血培养阳性患者中,发现病原菌以革兰阴性菌占比最高,为50.00%(6/12),其次是革兰氏阳性菌33.33%(4/12)与真菌16.67%(2/12);不同病原菌患者PCT水平经对比差异有统计学意义(P<0.05),且革兰氏阴性菌患者PCT水平高于革兰氏阳性菌与真菌的患者,差异有统计学意义(P<0.05)。结论PCT对诊断血流具有重要意义,并且快速简单,并不能准确确定病原体,可联合使用PCT与血培养,血培养弥补了PCT中的不足,从而可作为诊断血流感染最有价值检测方法。

时间分辨荧光免疫分析技术与牦牛肉兽药残留检测研究进展

时间分辨荧光免疫分析技术(Time-resolved fluorescence immunoassay.TRFIA)是一种以镧系元素或其微球作为标记物,测量标记物所发射荧光标记分析技术,其特点是灵敏度高且无放射性污染[1]。由于TRFIA技术有着传统方法所不具备的检测成本低,检测速度快等诸多优势,因此TRFIA技术为牦牛肉质安全检测开辟一条崭新的检测途径。该文就TRFIA技术及其在牦牛肉质安全检测应用进展做一简要综述。