人血清癌胚抗原的生物素-亲和素ELISA检测方法的建立

目的:建立检测血清癌胚抗原(CEA)的高灵敏度生物素-亲和素酶联免疫检测(BA-ELISA)方法。方法:亲和层析纯化得到CEA,免疫新西兰家兔制备多克隆抗体。将得到的抗体连接生物素和辣根过氧化物酶,在生物素化BSA包被的96微孔板上建立生物素-亲和素ELISA(BA-ELISA)。对这一体系的线性、灵敏度、特异度、稳定性、回收率等参数进行鉴定,并和常规ELISA、放射免疫检测以及化学发光法相比较检测了临床标本中的CEA浓度。结果:线性检测范围为(0.42～50)U/mL,检测结果表明,体系稳定性良好;灵敏度为0.42 U/mL,批内、批间差异均小于10.0%。该研究建立的方法与常规ELISA对比差异有统计学意义,与放射免疫检测对比差别无统计学意义。与化学发光法相比较,回归方程为:y=0.04825+0.99674x,r=0.994,差异无统计学意义。结论:建立的CEA的BA-ELISA检测方法易于操作、灵敏度高、价格低廉,适合应用于临床检测。     

地高辛的生物素—链霉亲和素免疫法测定方法的建立

目的：将生物素链霉亲和素免疫反应系统引入地高辛血药浓度监测中，准确地监测地高辛血药浓度。方法：将生物素与抗ＤＩＧＩｇＧ化学连接，ＤＩＧ通过ＨＳＡ与ＨＲＰ连接，建立了生物素链霉亲和素免疫反应系统测定地高辛的竞争反应模型。结果：以０５、１、２、３、５μｇ／Ｌ五个标准点建立了竞争反应曲线，同时将ＢＮＨＳＩｇＧ作了１：４００、１：８００、１：１６００、１：３２００的稀释，探讨了竞争反应曲线的规律性变化，运用流行的四参数曲线拟合法对竞争曲线作了拟合。同时做了方法学评价。结论：流行的生物素链霉亲和素技术可以引入被动的酶联免疫吸附法中，提高反应的灵敏度     

双抗体夹心生物素-亲和素ELISA法检测相思子毒素

目的 建立相思子毒素(abrin)的酶联免疫检测方法,为abrin临床诊断、中毒治疗、法医学鉴定等应用领域提供技术基础和参考依据.方法采用双抗体夹心生物素-亲和素ELISA法来检测微量abrin.结果该法检测abrin线性范围为0.125～31.25 μg/L,线性回归方程为y=0.52369X 0.51632(r=0.9816,P＜0.0001,n=9),检测限为0.125 μg/L.不同浓度蓖麻毒素(ricin)、葡萄球菌肠毒素(SEB)对检测结果基本无干扰,表明该法检测abrin具有很好的特异性.该法能用于abrin毒素污染水样、土样、食品、血液等模拟样品的分析,相对标准差为2.35%～4.14%,具有较好的重现性.结论 成功建立了夹心BA-ELISA法检测abrin,巧妙地将多克隆抗体的强富集能力、单克隆抗体的特异性以及生物素-亲和素系统的放大作用结合起来,达到了提高检测的灵敏度和特异性的目的 ,可适用于各种微量abrin样品的分析.     

人血清生长激素化学发光免疫分析方法的建立

建立链霉亲和素-生物素双抗体夹心化学发光免疫分析法(CLIA)测量人血清生长激素(GH),并进行临床应用检测.以链霉亲和素包被微孔板,生物素化一株GH单克隆抗体(McAb),辣根过氧化物酶标记另一株McAb,校准品与国家标准品进行溯源,建立GH CLIA法,进行性能参数测试,与国外试剂盒比对.结果表明本法线性范围为2....     

IgG3酶联免疫分析方法的建立及初步应用研究

目的:建立IgG3酶联免疫分析(ELISA)方法,探讨IgG3在炎症性疾病患者血清中的水平变化.方法:用兔抗人IgG3的多克隆抗体包被成固相酶标板,以识别结合待测标本中的IgG3,再用此多克隆抗体标记生物素(Bio-Ab),用亲和素标记辣根过氧化物酶(HRP-A),向固相酶标板中加入标准品或待测品,反应、洗涤后,加入Bio-Ab,反应、洗涤后,加入HRP-A,反应、洗涤后,酶标板上形成Ab-Ag-Ab-Bio-A-HRP复合物,加酶底物显色,用酶标仪在相应波长下测定光密度(OD值),根据标准曲线,计算出待测标本中的IgG3含量.结果:该法测定范围为(1.875～60)ng/ml,最低检出量1.5ng/ml,批内和批间误差分析＜8%和10%.用该法测得正常青年人血清IgG3含量为(12.91±4.25)ng/ml(n=64),风湿病患者为(11.23±4.64)ng/ml(n=64),肺部感染患者为(9.32±2.00)ng/ml(n=24),后两组血清IgG3水平均显著低于正常青年人(P＜0.05).结论:我们建立的IgG3 ELISA方法稳定、简便,特异性和灵敏度高,适于检测人血清IgG3水平的变化.     

IgG4酶联免疫分析的建立及初步应用

用兔抗人IgG4的多克隆抗体(IgG4Ab)包被成固相板,以识别结合待测标本中的IgG4。用生物素标记IgG4Ab得BioIgG4Ab;用亲和素标记辣根酶得HRPA。在已包被IgG4Ab的固相板中加入IgG4标准品(或待测样品),反应、洗涤后,加入BioIgG4Ab,反应、洗涤后,加入HRPA,反应、洗涤后,板上形成IgG4Ab—IgG4—BioIgG4Ab—HRPA复合物,加酶底物显色,用酶标仪在490nm波长测定OD值,作标准曲线,根据标准曲线,查出标本中IgG4含量。该法测定范围:2.5～200ng/mL,最低检出量2.1ng/mL,批内和批间CV分别8.4%和11.2%。测得血清IgG4含量:青年人(30名)为37.7±2.3ng/mL;30名献血员为42.7±9.9ng/mL,49例重症监护患者明显升高为71.2±9.2ng/mL;49例肺部感染患者明显降低为28.4±9.2ng/mL。结果表明该法稳定,灵敏度适于检出人血清IgG4水平。     

促甲状腺激素生物素-亲和素酶促化学发光免疫分析方法的建立

目的建立一种低成本、高灵敏度的检测血清促甲状腺激素(TSH)的生物素-亲和素酶促化学发光免疫分析方法。方法引入生物素-亲和素放大系统,一株抗TSH的单克隆抗体包被微孔板,另一株标记生物素,辣根过氧化物酶标记链亲和素,鲁米诺作为发光底物,采用夹心法,建立TSH酶促化学发光免疫分析方法。结果方法分析灵敏度为0.01mIU/L,批内变异系数为2.01%～5.40%,批间变异系数为2.96%～17.9%,回收率为90.6%～114.4%,高值促甲状腺激素血清样品经系列倍比稀释后,测定值与稀释度呈线性关系,相关系数大于0.99,与原子高科TSH免疫放射分析方法和罗氏电化学发光分析法的测定值具有良好的相关性。结论生物素-亲和素TSH酶促化学发光免疫分析方法具有成本低、灵敏度高、特异性高和稳定性好的特点,具有广阔的应用前景。     

C－反应蛋白生物素亲和素免疫酶标法的建立及其在人肝组织中的表达

Ｃ－反应蛋白生物素亲和素免疫酶标法的建立及其在人肝组织中的表达高艳琴，袁胜涛，胡锡琪，汪乃经Ｃ－反应蛋白（Ｃ－ｒｅａｃｔｉｖｅｐｒｏｔｅｉｎ，ＣＲＰ）主要由肝细胞合成，多存在于血液、胸水、腹水中，当机体处于感染、外伤及炎症活动期，ＣＲＰ的水平急骤上升...     

人禽流感酶联免疫诊断方法的建立及其初步应用

目的建立人禽流感H5抗体的酶联免疫检测方法（ELISA）。方法用ELISA法检测疑似高致病性禽流感患者及正常人群血清标本中H5IgG抗体。结果23例疑似高致病性禽流感患者血清样品中有4例H5IsG抗体阳性，阳性率为17．40％，与血凝抑制试验（HI）和微量中和试验（NI）相比较，三者符和率为100％。234例正常人群血清H5 IgG抗体检测均为阴性。结论建立的ELISA方法可用于高致病性人禽流感的血清样品初步筛查。     

人血清总甲状腺素（T_4）和总三碘甲腺原氨酸（T_3）生物素亲合素定量酶联免疫分析（BA－ELISA）

本文所介绍的Ｔ４－ＢＡ－ＥＬＩＳＡ及Ｔ３－ＢＡ－ＥＬＩＳＡ分析，其灵敏度已达到甚至超过了放射免疫分析的灵敏度，标准曲线范围与放射免疫分析相同。标准曲线的线性良好，血清稀释曲线、回收率及批内、批间变异系数均符合免疫分析的质量控制要求。操作简便、快速，保存期长，避免ＲＩＡ使用放射性同位素及保存期短的缺点。分别检测１２３例及１１４例正常人血清的总Ｔ４及总Ｔ３值，与有关文献报导的Ｔ４－ＲＩＡ、Ｔ３－ＲＩＡ的结果基本相符。用本方法测定甲亢及甲低病人血清总Ｔ４及总Ｔ３浓度，升高和降低与临床诊断相符合。     

人粒细胞弹性蛋白酶的酶免疫测定法及其临床意义

近几年来发现,中性粒细胞在多种疾病的发病学中有广泛的作用。粒细胞弹性蛋白酶(neutrophil elastase, NE)是粒细胞嗜天青颗粒中的一种作用范围广、水解能力强的蛋白水解酶,与急慢性炎症及多种组织损伤性疾病的发生有密切关系。我们建立了一种测量人血浆NE的酶免疫——生物素法,需血浆量为20μl。正常不吸烟成人血浆NE含量为33.7±6.7ng/ml((?)±SD);慢性阻塞性肺疾患病人为48.0±10.9ng/ml;皮肤化脓性感染病人为69.1±15.7ng/ml,系统性红斑狼疮病人为61.0±13.9ng/ml;而再生障碍性贫血病人仅12.3±5.3ng/ml,这些结果与正常人相比均有显著差异。作者认为;本方法特异性好,灵敏度可达毫微克水平,可望作为探讨NE在疾病发生中的作用及监测某些疾病变化的较好指标。     

An enzyme‐linked immunosorbent assay for heat‐shock protein 70 in plants

Procedures are described for the production of antibodies against heat‐shock protein 70 (HSP70) from plants and for the development of an enzyme‐linked immunosorbent assay (ELISA) for the antigen in crude plant extracts. Competitive and two‐site sandwich variants of the ELISA are compared. Results are presented for a preliminary analysis of HSP70 levels in control and heat‐shocked mung bean shoots.     

An enzyme‐linked immunosorbent assay for deoxynivalenol in wheat,utilizing novel hapten derivatization procedures

Polyclonal anti‐deoxynivalenol (vomitoxin) antisera was produced in rabbits. The immunogen was synthesized by using acetyl esterase to deacylate 3‐acetyldeoxynivalenol hemiglutarate and coupling the product to bovine serum albumin using the mixed anhydride reaction. The antiserum was very specific for deoxynivalenol in a microtitration plate ELISA that had a limit of detection of 90 pg per well. A simple, rapid extraction procedure was employed to enable the ELISA to be used for analysis of the deoxynivalenol in wheat.     

动物组织中盐酸克伦特罗残留ELISA检测试剂盒的研制

目的本研究采用直接竞争酶联免疫吸附试验(ELISA)对动物组织中盐酸克伦特罗(CBL)残留量进行快速筛选检测。方法将CBL与蛋白质载体偶联制备了CBL-BSA和HRP-CBL结合物,以CBL-BSA免疫实验兔获得特异性抗体,并成功建立了CBL残留直接竞争ELISA检测方法及检测试剂盒。结果检测线性范围0.1-62.5 ng/mL,检测低限为0.10 ng/mL;尿、肝、肉和饲料的加标回收率为60.3%-115.0%;与英国Randox试剂盒对比测试结果无显著差异;重现试验变异系数为3.4%-7.2%;试剂盒贮藏在2-8℃5个月和30℃7 d质量稳定。结论本试剂盒具有操作简便、快速、灵敏等特点。     

A highly sensitive enzyme-linked immunosorbent assay for copper(II) determination in drinking water

Monoclonal antibodies against Cu(II)–EDTA were produced by using an immunogen of Cu(II) chelate conjugated to keyhole limpet hemocyanin (KLH) via a bifunctional chelator, 1-(4-isothiocyanobenzyl)ethylenediamine-N,N,N′,N′-tetraacetic acid (ITCBE). Stable hybridoma cell lines were generated with immunisation in mice followed by cell fusion. A monoclonal antibody with high affinity and specificity for Cu(II)-EDTA but not metal-free EDTA was produced from a selected hybridoma cell line (B11) and used to develop a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) for detection of Cu(II). The assay conditions, including the antigen coating, plate blocking and buffer pH, and ionic strength were examined for optimal assay performance. The optimised assay showed less than 3.9% cross-reactivities for Zn(II) and Co(II) and no cross-reactivities for other tested metal ions. The assay showed high sensitivity for Cu(II) with an IC₅₀ of 3.9 ng/mL, a detection limit of 0.24 ng/mL and a detection range of 0.67–29 ng/mL. Recoveries from drinking water were determined to be in the range of 94.4–117.2%.     

Indirect enzyme‐linked Immunosorbent assay for detection of cow's milk in goat's milk

An indirect enzyme‐linked immunosorbent assay (ELISA) has been developed for the specific detection of cow's milk (1–25%) in goat's milk. The test uses polyclonal antibodies raised in rabbits against bovine whey proteins (BWP). The anti‐BWP antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized BWP. The anti‐BWP antibodies were biotinylated and rendered cow's milk specific by mixing them with lyophilized ovine and caprine whey proteins. Streptavidin‐peroxidase was used to detect the biotinylated anti‐BWP antibodies bound to bovine milk proteins immobilized on 96‐well plates. The colour developed by the subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of goat's milk containing variable amounts of cow's milk.     

An enzyme‐linked immunosorbent assay for cellulase (from trichoderma viride) in animal feed

Cellulase can be added to animal feed mixture for the improvement of nutrient conversion. An enzyme‐linked immunosorbent assay has been developed to check the concentration of added enzyme. The IgG fraction from rabbit immune serum against cellulase from Trichoderma viride was labelled with horseradish peroxidase by the glutaraldehyde method. The conjugate (IgG‐Px) was characterized by gel chromato‐graphy on a Superose 12 column. The assay was optimized with respect to the amount of IgG‐Px applied and the reaction conditions. The method developed was utilized for the assay of feed granules supplemented by cellulase preparations. It is possible under the conditions described to determine added cellulase up to a concentration of 24.0 μg/g of the feed mixture.     

Immunoglobulin responses to echovirus type 11 by enzyme linked immunosorbent assay: Single-serum diagnosis of acute infection by specific IgM antibody

An indirect solid-phase enzyme-linked immunosorbent assay (ELISA) for the determination of specific IgM and IgG antibodies to echovirus type 11 in a single dilution of serum was developed using partially purified echovirus type 11 bound to microplates. Whole serum was used for IgG antibody but prior to assaying for IgM antibody interfering IgG was removed by ion exchange chromatography. The ELISA for echovirus type 11 IgG antibody was a more sensitive, rapid, technically easier and less costly alternative to the neutralisation test. With the IgG ELISA 12 of 132 sera (10.6%) known to contain enterovirus antibodies other than echovirus type 11 were positive but it could not be determined to what extent this was due to the greater sensitivity of the ELISA or cross-reactions. The IgM ELISA was even more sensitive than the IgG ELISA with acute sera, and showed a reactivity in 4 of 36 sera (11.1%) with no detectable echovirus type 11 neutralising antibodies. Echovirus type 11 IgM antibody was detected in all sera collected after the first week of infection and up to 30 days after infection. However, it was only detected in 58% of sera collected during the first week after onset thus limiting its use for rapid diagnosis. The echovirus type 11 IgM ELISA appears to have considerable laboratory diagnostic potential when a rising antibody level cannot be demonstrated in paired sera or when virus is not cultured.     

桥式生物素—亲合素标记提高藻胆蛋白免疫荧光法灵敏度的研究

目的 提高藻胆蛋白免疫荧光法检测的灵敏度。方法 在藻胆蛋白免疫荧光法（PBPIFA）的检测流程中,引入桥式亲合素-生物素系统（BRAB）,称为BRAB-PBPIFA,用ELISA法与藻胆蛋白免疫荧光直接法和桥式BAS法,对定值的标准质控抗原HBsAg样品及500份血清样品同时进行测定,结果 BRAB应用于藻胆蛋白免疫荧光法中,能显著提高该法对低抗原含量样品的检出,对弱阳性血清的检出率可提高1.5倍,无蛋白封闭试剂Tween20在BRAB-PBPIFA中具有较好的封闭效果,结论 BRAB标记可提高藻胆蛋白免疫荧光法检测的灵敏度。     

Development of heterologous enzyme linked immunosorbent assay for dehydroepiandrosterone in serum

Homologous and heterologous combinations of enzyme conjugate with immunogen in steroid enzyme immunoassay (EIA) influence labeled steroid recognition by an antibody that affects the sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) enzyme linked immunosorbent assay (ELISA), antibodies were generated against dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) and dehydroepiandrosterone-7-carboxymethyloxime- bovine serum albumin (DHEA-7-CMO-BSA). Two dehydroepiandrosterone (DHEA) horse radish peroxidise (HRP) enzyme conjugates were prepared using two dehydroepiandrosterone derivatives (DHEA-17-CMO and DHEA-7-CMO). Four combinations of homologous and heterologous assays were evaluated. The use of heterologous combination improved the sensitivity of the assay.