氯霉素磁分离酶联免疫(MEIA)检测方法的建立

目的:采用磁分离酶联免疫分析法(MEIA)建立定量检测氯霉素(CAP)的一种高灵敏度方法.方法:采用免疫竞争法,用异硫氰酸荧光素(FITC )标记抗CAP抗体,碱性磷酸酶(AP)标记CAP,以羊抗FITC抗体包被的免疫磁珠作为固相分离载体,单磷酸酚酞做显色底物,建立检测CAP的MEIA法.结果:成功建立了检测CAP的MEIA法,检测时间为40 min,检测灵敏度为0.03μg/L,线性范围为0.1～8.1μg/L,批内变异系数(CV) 3.9%-5.3%,批间CV 4.8%-8.1%,回收率为97%～101.5%,与国标方法液相色谱一串联质谱法的检测结果对比,相关系数r=0.9817.结论:检测CAP的MEIA法的灵敏度及准确率高、检测时间短,为食品中氯霉素残留的监测提供了一种新的免疫分析方法.     

磁分离酶联免疫法检测IL-4方法的建立

建立磁分离酶联免疫法定量检测人白细胞介素4(IL-4)的方法.用异硫氰酸荧光素(FITC)和碱性磷酸酶(AP)分别标记识别不同表位的两种抗IL-4单克隆抗体(mAb), 用抗FITC多抗或mAb包被免疫磁珠.结果显示: 检测标准品浓度在0-3ng/mL范围内线性关系良好, 敏感性达10pg/mL, 平均回收率为101.7%,批内批间CV均为3.6%,与ELISA比较相关系数为0.9124.此法所需样本量少, 方法稳定, 敏感性高和特异性强, 又无放射性污染, 在临床检验和基础研究中有较大应用价值.     

微粒子化学发光免疫测定促甲状腺素敏感性方法的建立与应用

目的 :建立一种低成本、高敏感性检测血清促甲状腺激素(TSH)的微粒子化学发光免疫分析 (CLEIA)方法。方法 :采用碱性磷酸酶标记的抗TSHβ亚单位特异性单克隆抗体 (mAb) ,与FITC偶联的抗TSHα亚单位的另一mAb配对 ,构成双抗体夹心免疫结合 ,用抗FITC多抗免疫磁珠做固相分离载体 ,用金刚烷胺CSPD为发光底物的非均相免疫分析法。结果 :方法灵敏度 4 μIU/L ,批内变异系数 (CV)平均为7.4 5 % ,批间CV平均为 10 .4 5 % ,回收率为 91.4 %～ 10 2 .4 %。将实际样品测定与ACS 180  系统检测的结果相比较有较好的相关性。结论 :微粒子化学发光定量测定TSH的成本低、灵敏度及特异性高和稳定性好 ,具有广阔的应用前景     

人绒毛膜促性腺激素游离β亚单位磁分离酶联免疫方法的建立

目的建立一种新的测定人绒毛膜促性腺激素游离β亚单位(hCGβ)磁分离酶联免疫分析法(MEIA)。方法用识别不同表位的两株单克隆抗体(mAb),一株mAb用FITC标记,另一株用碱性磷酸酶(AP)标记;以偶联羊抗FITC抗体的磁珠作为固相,以酚酞单磷酸酯溶液为底物,建立测定hCGβ磁分离酶免疫测定法。结果本MEIA法的灵敏度为0.1IU/L,批内CV小于8.5%,批间CV小于14%,平均稀释回收率为92.5%;与促黄体生成激素(leuteinizinghormone,LH)、促甲状腺激素(thyroidstimulatinghormone,TSH)和促卵泡成熟激素(folliclestimulatinghormone,FSH)均无交叉反应,与完整的hCG(1000IU/L)的交叉反应为1.2%,有效期不低于14个月。结论本方法的性能优于现有的放射免疫(RIA)和ELISA试剂盒,接近进口同类试剂的水平,有助于为市场提供一种高质量而价廉的hCGβ测定试剂盒。     

血清真胰岛素化学发光免疫分析方法学研究

建立一种高灵敏度真胰岛素临床常规检测方法.采用两株特异的抗胰岛素单抗体,分别抗胰岛素肽链上两个不同的抗原决定簇,用一株制备固相抗体,另一株标记碱性磷酸酶C,以金刚烷衍生物为发光底物建立化学发光免疫分析(CLIA)法.结果表明本方法灵敏度为0.06μIU/mL,标准曲线量程为1.0～150μIU/mL,批内和批间CV分别为5.0%、7.8%,平均回收率为94.4%.分别测定了男、女成人空腹血清各100份,男性实测范围0.52～11.23μIU/mL,平均值为3.86μIU/mL,女性实测范围为0.75～10.66μIU/mL,平均值为3.62μIU/mL.真胰岛素CLIA法简便快速,能真实反应血清胰岛素的水平,对临床糖尿病的诊疗和病理生理研究具有实用价值.     

C-肽双标记液相IRMA方法的实验研究

本文探讨应用两株高特异性配对C-P McAb分别标记125I和异硫氰酸荧光素(FITC),采用抗FITC磁性微粒子固相抗体作为分离剂,建立血清C-P IRMA检测法。结果表明,本法标准曲线范围为0.125~4.0pmol/mL,灵敏度为0.06 pmol/mL,批内批间CV分别为5.6%和8.7%,回收率为92.1%~96.0%,和胰岛素交叉反应为零。各项技术参数表明本法操作简便快速,为今后开发肽类的IRMA检测提供了特异性更高的手段。     

化学发光免疫分析血清C肽方法建立

双抗体夹心一步法建立人C肽(C-peptide) 化学发光免疫分析(CLIA)方法.方法可测范围0.15～15ng/mL,灵敏度0.02ng/mL,批内、批间变异系数(CV)分别为4.2%～6.5%和6.8%～9.3%,与胰岛素无交叉反应,与胰岛素原的交叉反应率为7.3%.本方法与国外同类试剂盒同时检测40份C-肽释放试验血清,相关系数为0.9709.本方法各项指标均满足临床检测要求,达到国外同类产品水平.     

FITC与抗-FITC系统检测促黄体生成素的应用评价

目的 使用抗-异硫氰酸荧光素(FITC)固相包被板建立促黄体生成素(LH)的酶联免疫(ELISA)检测方法.方法用FITC及辣根过氧化物酶(HRP)分别标记两株抗-LH单克隆抗体,建立一步检测LH的ELISA检测方法,并与经典的双抗体夹心法进行了方法学对比评价.结果 FITC与抗-FITC系统检测LH,在2~50mIU/mL的校准曲线范围内相关系数0.9968,分析内精密度为7.6%,分析间精密度为7.02%,热稳定性下降18.5%,与双抗体夹心法相当,空白检测限为0.15mIU/mL,优于双抗体夹心法,钩状效应(HOOK效应)比双抗体夹心法差.与贝克曼检测结果回归方程Y=0.970X+0.614,相关系数r=0.975.结论 成功建立基于FITC与抗-FITC系统的ELISA检测方法,与双抗体夹心法相比,两种方法均能满足临床检测的需要.     

促甲状腺素化学发光免疫定量检测试剂盒的研制

目的:研制人血清中促甲状腺素(TSH)的化学发光免疫检测试剂盒.方法:采用辣根过氧化物酶标记的抗TSHβ亚单位特异性单克隆抗体(mAb),与固相包被的抗TSHα亚单位的另一mAb配对,以鲁米诺作为底物,采用两步法建立了双位点夹心法人血清TSH的化学发光免疫定量分析法.结果:该方法灵敏度为0.0788 mIU/L,批内CV平均为4.7%,批间CV平均为8.4%,平均回收率为93.05%.该检测与LH、FSH和HCG无交叉反应.部分实验样品的测试结果显示该试剂与国外同类试剂(雅培architect i2000)的测定结果明显相关(r＝0.984).结论:该方法具有较高的灵敏度、重复性和准确度,与其他同类产品相比简便、快捷、成本低,适于临床检测和科研应用.     

链霉亲和素-生物素化学发光免疫分析血清胰岛素方法建立及临床应用

目的：建立链霉亲和素-生物素双抗体夹心一步法测量人胰岛素化学发光免疫分析方法,并进行临床应用检测。方法：以链霉亲和素包被微孔板,生物素化一株单克隆抗体,辣根过氧化物酶标记另一株单克隆抗体。校准品与胰岛素国家标准品进行溯源,建立免疫分析方法,进行性能参数测试。收集40例正常人、12例1型、29例2型糖尿病人血清,统计整理参考值,并与国外同类试剂盒进行对比。结果：该方法回收率94.13%,线性范围（2～200）μIU/ml,灵敏度0.05μIU/ml,批内、批间变异系数（CV）分别为2.87%～7.06%和3.24%～9.14%,与牛胰岛素、猪胰岛素、人前胰岛素原、胰岛素样生长因子Ⅰ交叉反应率分别为27.2%、19.7%、0.16%、0.07%,与C肽、胰高血糖素、生长抑素无交叉反应。本法与国外同类试剂盒同时检测40份正常人葡萄糖耐量试验血清,r为0.9869,本法37℃7d热稳定性良好,建立一组正常人葡萄糖耐量参考值。结论：本法各项指标均满足临床检测要求,达到国外同类产品水平,反应快速,操作简单,便于临床检验应用。     

Evaluation of a solid-phase monoclonal antibody-based enzyme immunoassay for insulin in human serum

A 'sandwich-type' enzyme immunoassay for the measurement of serum insulin is described in which a monoclonal antibody-alkaline phosphatase conjugate and an antibody-immobilized polystyrene solid phase are used. Serum samples of 50 microliters can be analyzed and the enzyme immunoassay is as sensitive as the conventional radioimmunoassay for insulin. The results obtained with ELISA correlate well with those of the radioimmunoassay (r = 0.9844) and the between-assay and within-assay coefficients of variation are less than 15% over the useful ranges of the assay (2-200 microIU/ml). The sensitivity is 2 microIU/ml and this can be increased by longer incubation times. The crossreaction with porcine insulin is 45%, with bovine insulin 30% and with human proinsulin 20%.     

Immunoradiometric assay (IRMA) for human follicle stimulating hormone (FSH) and luteinizing hormone (LH) using common avidin solid phase

This paper describes the use of avidin-biotin interaction as an affinity system, wherein avidin immobilized magnetizable particles (cellulose) are used as a common separation system in immunoradiometric assay (IRMA) for hormones of the human reproductive system, human follicle stimulating hormone (FSH), and luteinizing hormone (LH). Biotinylated probe was prepared by biotinylation of specific monoclonal antibody for respective antigen using the caproyl derivative of biotin N-hydroxysuccinimide. The detector antibody for the respective antigen was radiolabelled with 125I by a chloramine-T oxidation method and purified by gel filtration. In the IRMA procedure, standard/sample, respective biotinylated, and radiolabelled antibody as a single reagent, and avidin solid phase were added simultaneously to the assay tubes. After incubation for 3 h with shaking, the bound complex was quantitated for its radioactivity associated with the common avidin solid phase. Results showed that the developed assay protocol is applicable to IRMA of FSH and LH with good precision (intra and inter assay CV less than 8% and 11%, respectively), good assay range (0-200 mIU/mL) and analytical recovery (87-110%). The assay could detect 0.5 mIU/mL and 0.9 mIU/mL of FSH and LH, respectively, and showed good correlation with commercially available kits (FSH y = 0.98x + 0.21 and LH y = 0.99x + 0.18).     

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。     

Development of chemiluminescent enzyme immunoassay for the determination of malachite green in seafood

An indirect competitive chemiluminescent enzyme immunoassay method was developed for screening malachite green (MG). Assay conditions, including the concentration of coating, antibody dilution, incubation time, dilution buffer, ionic strength and pH, were optimised. Under the optimised conditions, coating antigen concentration was 125 ng/mL; and dilution fold of antibody was 40,000. The 50% inhibition values of 0.22 ng/mL for MG were achieved with a limit of detection of 0.01 ng/mL, the linear range was from 0.03 ng/mL to 3.27 ng/mL. The assay showed a little cross-reactivity of 3.4%, 2.7% and 1.0% with methylene blue, brilliant green and crystal violet, respectively, and negligible cross-reactivity with other analogues of MG. The developed method has been used to quantify MG in seafood samples. The recovery of MG ranged from 82.43% to 108.0% at four concentrations (0.1, 1, 3 and 5 ng/mL), and the coefficients of variation were less than 13%. A comparison results between the high-performance liquid chromatography and the developed assay showed good relativity. The results indicated that the assay was a sensitive and stable method for screening MG.     

Performance characteristics of a novel magnetic‐particlebased enzyme‐linked immunosorbent assay for the quantitative analysis of atrazine and related triazines in water samples

The performance characteristics of a magnetic‐particle‐based solid‐phase enzyme‐linked immunosorbent assay (ELISA) requiring no sample preparation for the quantification of atrazine and related triazines in groundwater samples is described. Water samples and HRP‐labeled atrazine are incubated with the antibody‐coupled solid phase for 15 min. A magnetic field is then applied to the solid phase to wash and remove free HRP‐labeled atrazine and to ensure the removal of potential interfering substances. After 20 min of color development using hydrogen peroxide/TMB, the reaction is stopped by the addition of acid. Photometric analysis of the final colored reaction was made using a specially designed microprocessor‐controlled photometer with extensive data reduction capability. The assay was found to be free of interferences when challenged with high concentrations of inorganic ions and a wide variation in sample pH. Sensitivity of the assay was 50 ppt based on 95% B/B₀. Assay precision demonstrated coefficients of variation of less than 10%. The ELISA compared favorably with GC/MS measurements. The broad antibody specificity allows for the detection of a majority of triazines.     

Determination of zearalenone in corn based on a biotin-avidin amplified enzyme-linked immunosorbent assay

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed to detect zearalenone (ZEN) residues in corn. The concentration of coating antigen, biotinylated monoclonal antibody specific to ZEN, avidin-horseradish peroxidase and reaction time in the system of BA-ELISA method were optimised. Under the optimum conditions, a good linear relationship between binding ratio and ZEN concentration in the range of 0.54 to 7.99 ng/mL was obtained. The regression equation was y=?21.26 ln(x)+66.89 (R²=0.9989). The limit of detection and limit of quantification were 0.35 ng/mL and 0.812 ng/mL. The IC₅₀ was calculated to be 2.071 ng/mL. In comparison with the traditional ELISA method, the sensitivity of BA-ELISA method developed has been elevated by six times. The recovery and coefficient of variation with the spiked corn samples were in the range of 86.6% to 93.7% and less than 7.8%, respectively. It promises a bright future for the sensitive and rapid detecting method of ZEN residues in corn.     

An enzyme immunoassay for rat prolactin: application to the determination of plasma levels

Pure acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus has been covalently coupled to rat prolactin using the heterobifunctional reagent: N-succinimidyl-4 (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). This conjugate was used as a tracer in a competitive enzyme immunoassay using a rabbit antiserum, raised against rat prolactin, as first antibody. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody. This second antibody solid phase ensured separation of bound and free moieties of the tracer during the specific immunoreaction. The total reaction volume was 150 microliters. Each component (tracer, antiserum and standard) was added in a volume of 50 microliters. The sensitivity of the assay was good since calculation indicated a detection threshold of 25 pg (0.5 ng/ml) and a B/Bo 50% value of 220 pg (4.4 ng/ml). Intra-assay variation was better than 10% over a wide range (135 to 2500 pg) with an optimum of 4% at 300 pg. The inter-assay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 8 to 1000 ng/ml. The good parallelism observed between the standard curve and sample dilution curves, and recovery experiments, indicated that direct assay is possible. This was confirmed by molecular sieve fractionation of plasma samples. The reliability of the assay was confirmed by good correlation with conventional radioimmunoassay (r = 0.996, slope = 0.978).     

Immunoradiometric Assay (IRMA) for Human Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) Using Common Avidin Solid Phase

Abstract

This paper describes the use of avidin-biotin interaction as an affinity system, wherein avidin immobilized magnetizable particles (cellulose) are used as a common separation system in immunoradiometric assay (IRMA) for hormones of the human reproductive system, human follicle stimulating hormone (FSH), and luteinizing hormone (LH). Biotinylated probe was prepared by biotinylation of specific monoclonal antibody for respective antigen using the caproyl derivative of biotin N-hydroxysuccinimide. The detector antibody for the respective antigen was radiolabelled with ¹²⁵I by a chloramine-T oxidation method and purified by gel filtration. In the IRMA procedure, standard/sample, respective biotinylated, and radiolabelled antibody as a single reagent, and avidin solid phase were added simultaneously to the assay tubes. After incubation for 3 h with shaking, the bound complex was quantitated for its radioactivity associated with the common avidin solid phase. Results showed that the developed assay protocol is applicable to IRMA of FSH and LH with good precision (intra and inter assay CV less than 8% and 11%, respectively), good assay range (0–200 mIU/mL) and analytical recovery (87–110%). The assay could detect 0.5 mIU/mL and 0.9 mIU/mL of FSH and LH, respectively, and showed good correlation with commercially available kits (FSH y = 0.98x + 0.21 and LH y = 0.99x + 0.18).     

Performance characteristics of immunoenzymatic allergosorbent testing for total and specific immunoglobulin E

Paper disc-based solid phase radioimmunoassays are widely used in vitro diagnostic test kits for measuring total IgE (Phadebas PRIST) and specific IgE antibodies (RAST). Recently, these kits have been modified by the substitution of an enzyme-linked immunosorbent assay detection system (Phadezym PRIST/RAST). We studied the performance characteristics of Phadezym PRIST and RAST kits. Phadezym PRIST was sensitive to 0.5 IU/mL IgE. Reproducibility was excellent in the range of 5 to 200 IU/mL IgE and adequate in the range of 5 to 1000 IU/mL using 1:10 serum dilutions (average inter-assay coefficient of variation = 19%). Phadezym RAST was specific, but sensitivity was limited by absorbances in the RAST class 1/0 range indistinguishable from background values. Average inter-assay coefficient of variation was 29% for the semi-quantitative 'Phadezym RAST Unit' (PRU) reporting system. We modified the test kit procedure by disc incubations in microtiter plate wells with rotational agitation and use of ELISA-dedicated spectrophotometer and computer software. These microplate accelerated computerized assays ('MacPRIST' and 'MacRAST') were shown to perform similarly to the conventional Phadezym procedures with advantages in speed, ease, and handling of data.