ELISA法和化学发光法对血清中HIV-1HIV-2抗体、梅毒抗体和丙肝抗体的检测意义

目的探讨分析ELLSA法(酶联免疫吸附法)和化学发光法对血清中HIV-1/HIV-2抗体、梅毒抗体和丙抗体的临床检测意义。方法将382份脐血作为研究分析对象,分别采用酶联免疫吸附和化学发光法对所选标本进行检测,包含丙肝抗体、梅毒抗体、HIV-1/HIV-2抗体检测。结果采用ELLSA检测后HIV-1/HIV-2抗体、梅毒抗体、丙肝抗体阳性率分别为0.52%、0.79%、1.05%。采用化学发光法检测HIV-1/HIV-2抗体、梅毒抗体、丙肝抗体3种检测的阳性率分别为0.79%、1.05%、1.31%。标准抗体品梯度稀释后再进行检测,将此3种抗体稀释程度为10 pg/m L,三组抗体采用化学发光法进行检测,其结果均为阳性,采用酶联免疫吸附方式进行检测的只有梅毒抗体呈阳性,其余两项均为阴性。结论化学发光法作为临床进行血清中HIV-1/HIV-2抗体、梅毒抗体、丙肝抗体检测的首选方案,此检测方式具有更高的灵敏性。     

红细胞标记抗体的化学发光免疫分析

用红细胞代替辣根过氧化物酶作为双抗体夹心免疫分析中第二抗体的标记物, 建立了一种红细胞标记抗体的免疫化学发光测定乙型肝炎病毒表面抗原的新方法. 在免疫反应完成后, 结合了抗原-抗体免疫复合物的致敏红细胞在低渗溶液中溶血, 释放出血红蛋白. 基于血红蛋白对鲁米诺-H2O2体系化学发光具有催化作用的原理, 采用化学发光法测定血红蛋白含量. 测得的血红蛋白发光强度与待测抗原浓度呈线性关系. 采用这种方法可检测出0.5 ng/mL的乙型肝炎病毒表面抗原. 将该方法与酶联免疫吸附分析(ELISA)结合起来对乙型肝炎患者血清乙肝病毒表面抗原(HBsAg)进行检测, 两者符合率均为97%, 表明本法具有良好的灵敏度和特异性, 可用于临床标本测试.     

流动注射化学发光免疫分析研究I. 血清中乙型肝炎表面抗原的测定

我们首次以键合有抗体的多孔玻璃作为固相免疫分析的免疫反应器, 以化学发光作为最终检测手段, 建立了一种新的、高效率的免疫分析技术-流动注射化学发光免疫分析技术。实验表明: 采用该技术可使单次测定时间从ELISA(Enzyme-linked Immunosorbent Assay)法的二十多小时降至二十分钟, 且所有操作均可在微机控制下自动完成。用该方法对人血清中乙型肝炎表面抗原的测定结果与ELISA法所得结果一致, 对同一样品连续九次测定的相对标准偏差为7.2%。因此, 该方法具有自动化程度高、分析速度快、稳定性好的优点。     

流动注射化学发光免疫分析研究I. 血清中乙型肝炎表面抗原的测定

我们首次以键合有抗体的多孔玻璃作为固相免疫分析的免疫反应器, 以化学发光作为最终检测手段, 建立了一种新的、高效率的免疫分析技术-流动注射化学发光免疫分析技术。实验表明: 采用该技术可使单次测定时间从ELISA(Enzyme-linked Immunosorbent Assay)法的二十多小时降至二十分钟, 且所有操作均可在微机控制下自动完成。用该方法对人血清中乙型肝炎表面抗原的测定结果与ELISA法所得结果一致, 对同一样品连续九次测定的相对标准偏差为7.2%。因此, 该方法具有自动化程度高、分析速度快、稳定性好的优点。     

偶合反应化学发光酶免疫分析研究: I. HRP及其标记物的化学发 光测定

本文将辣根过氧化物酶(HRP)催化过氧化氢氧化KI生成I~2的反应与Luminol-I~2的化学发光反应相偶合,提出了测定HRP及其抗原、抗体标记物的高灵敏度的化学发光法。检测下限为7pg,线性范围为10-6000pg。通常使用的HRP直接催化Luminol-H~2O~2化学发光法灵敏度高10-100倍;克服了固相吸附法直接测定HRP标记物的缺陷,提高了测定的选择性。文中还对该偶合反应的机理及其动力学特性进行了研究。     

偶合反应化学发光酶免疫分析研究: I. HRP及其标记物的化学发 光测定

本文将辣根过氧化物酶(HRP)催化过氧化氢氧化KI生成I~2的反应与Luminol-I~2的化学发光反应相偶合,提出了测定HRP及其抗原、抗体标记物的高灵敏度的化学发光法。检测下限为7pg,线性范围为10-6000pg。通常使用的HRP直接催化Luminol-H~2O~2化学发光法灵敏度高10-100倍;克服了固相吸附法直接测定HRP标记物的缺陷,提高了测定的选择性。文中还对该偶合反应的机理及其动力学特性进行了研究。     

偶合反应化学发光酶免疫分析研究 Ⅰ.HRP及其标记物的化学发光测定

本文将辣根过氧化物酶(HRP)催化过氧化氢氧化KI生成I_2的反应与Lumnol-I_2的化学发光反应相偶合,提出了测定HRP及其抗原、抗体标记物的高灵敏度的化学发光法.检测下限为7pg,线性范围为10—6000pg.较通常使用的HRP直接催化Luminol-H_2O_2化学发光法灵敏度高10—100倍;克服了固相吸附法直接测定HRP标记物的缺陷,提高了测定的选择性.文中还对该偶合反应的机理及其动力学特性进行了研究.     

合并血浆中病毒标记检测临界值的确定分析

确保合并血浆检测结果的判定准确可靠,能够有效保证合并血浆的病毒安全性,对合并血浆乙型肝炎病毒表面抗原、丙型肝炎病毒抗体、人类免疫缺陷病毒抗体检测时不同厂家检测试剂的临界值进行确定。(1)使用双抗体夹心酶联免疫法对合并血浆HBsAg和HIV-1/HIV-2抗体的检测临界值进行确定。(2)使用酶联免疫法检测合并血浆HCV抗体的临界值进行确定。经检测和计算,两个厂家检测试剂的检测临界值系数分别为乙型肝炎病毒表面抗原23.398%和26.845%、丙型肝炎病毒抗体9.012%和16.481%、人类免疫缺陷病毒抗体20.025%和23.424%。     

板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125

在传统的板式化学发光免疫分析法和管式磁颗粒化学发光免疫分析法基础上,建立了人血清中糖类抗原125(CA125)的板式磁颗粒化学发光免疫分析方法.该方法以磁性微粒子作为分离固相,96孔板为反应容器,辣根过氧化物酶(HRP)催化H2O2-luminol化学发光体系作为检测体系.本法测定CA125的检测灵敏度可达2.0U/mL,线性范围为0～400U/mL.与常用的包被板化学发光免疫分析方法对比,该方法检测范围宽.与管式磁颗粒化化学发光法比较,其分析灵敏度与精密度高、线性范围、分析通量以及分析成本方面均显示了很好的优越性.采用该方法对人血清中CA125进行测定并与罗氏全自动电化学发光系统的测值结果进行了比对,两者显示了良好的相关性.     

纳米磁性微球免疫伏安法测定乙肝表面抗原

采用化学键合法将乙肝抗体固化在自行制备的纳米磁性高分子功能微球表面 ,利用免疫夹心反应原理 ,捕获溶液中的乙肝表面抗原和标记有辣根过氧化物酶的乙肝第二抗体 .在外加磁场的作用下 ,抗体抗原结合物从样品溶液中分离 ,在含有邻氨基苯酚和过氧化氢的底液中 ,生成具有电活性的化合物 3 氨基吩呃嗪 ,用示差脉冲伏安法进行测定 .响应电流与乙肝表面抗原浓度分别在 0 2～ 1 0和 1 0～ 5 0 0ng·mL-1成线性关系 ,检出限达 0 0 60ng·mL-1.采用本方法检测血清中乙肝表面抗原 ,灵敏度大大高于目前临床采用的酶联免疫吸附法 .     

Measurement of N-acetylneuraminic acid in human serum and urine by high performance liquid chromatography with chemiluminescence detection.

A highly sensitive method for the determination of N-acetylneuraminic acid in human serum and urine is investigated. This method employs high performance liquid chromatography with chemiluminescence detection. N-Acetylneuraminic acid, released by hydrochloric acid hydrolysis of serum and urine, and N-glycolylneuraminic acid (internal standard) are converted into chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for alpha-keto acids. The derivatives are separated within 35 min on a reversed phase column, TSKgel ODS-120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for N-acetylneuraminic acid is 9 fmol (signal-to-noise ratio = 3). This sensitivity permits precise determination of N-acetylneuraminic acid in 10 nL of serum or 50 nL of urine. The method is applied to the determination of the N-acetylneuraminic acid in human sera from normal subjects and cancer patients and in normal urine.     

偶合化学发光法测定血清中亚硝酸盐

采用正交试验法建立了Ｌｕｍｉｎｏｌ－ＮＯ－２－Ｋ４［Ｆｅ（ＣＮ）６］偶合发光体系测定血清ＮＯ－２的方法。本法测定ＮＯ－２的线性范围为９．０×１０－６～１．０×１０－３μｇ／Ｌ,检出限３．５×１０－６μｇ／Ｌ。对１．０×１０－４μｇ／ＬＮＯ－２连续测定１１次的相对标准偏差为２．３％。用于血清中亚硝酸根的测定,结果满意。     

Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immobilized reagents technology

A novel chemiluminescence (CL) flow sensor for the determination of uric acid in human urine and serum has been developed by using controlled-reagent-release technology. The reagents involved in the chemiluminescence (CL) reaction, luminol and periodate, are immobilized on anion-exchange resin packed in a column. After injection of water, chemiluminescence generated by released luminol and periodate in alkaline media is inhibited in presence of uric acid. By measuring the decreased chemiluminescence (CL) intensity the uric acid is sensed. The decreased response is linear in the 5.0-500.0 ng mL(-1) range, with a detection limit of 1.8 ng mL(-1). The flow sensor showed remarkable operational stability and could be easily reused for over 80 h with sampling frequency of 100 h(-1). The proposed sensor was applied to the determination of uric acid in human urine and serum, and monitoring metabolic uric acid in human urine with RSD less than 3.0%.     

Application of chemiluminescence immunoassays for steroid hormones in clinical endocrinological investigations in women

Immunoassays based on chemiluminescence for the measurement of serum and plasma steroids (estradiol, estriol, progesterone, testosterone, and cortisol), urinary steroid conjugates (estrone-3-glucuronide, estriol-16α-glucuronide and pregnanediol-3α-glucuronide) and peptide hormones (choriogonadotropin and luteinizing hormone) are surveyed briefly. These immunoassays are simple, robust and valid alternatives to radioimmunoassay. Homogeneous procedures and recent solid-phase assays based on purified specific antibodies, covalently coupled to polymer beads are discussed. Some new results are presented for solid-phase chemiluminescence immunoassays: estradiol is quantified in extracts of serum by using a monoclonal antibody to estradiol with estradiol-6-carboxymethyloxime-aminobutylethyl isoluminol as the marker ligand, and progesterone is quantified in unextracted serum by using a polyclonal antibody to progesterone, progesterone-11-hemisuccinyl-aminobutylethyl isoluminol as the marker ligand, and danazol (17α-pregna-2,4-dien-20-yno[2,3-d]-isoxazol-17-ol) to displace progesterone from serum binding-proteins. Their clinical utility is demonstrated.     

高效液相色谱化学发光检测法测定氨基酸

本文根据铜与氨基酸组成的１：１不饱和络合物对Ｌｕｍｉｎｏｌ－Ｈ２Ｏ２化学发光体系的催化活性，设计了一种新型氨基酸高效液相色谱化学发光检测器，即在色谱柱后安装一个氢氧化铜柱，从柱后流出的氨基酸通过氢氧化铜柱时，产生配合溶解形成氨基酸－铜（１：１）络合物，进行化学发光检测，１４种常见氨基酸的检测限均在ｐｍｏｌ级，已用于血清、尿和啤酒中氨基酸的测定。     

Gold(III) enhanced chemiluminescence immunoassay for detection of antibody against ApxIV of Actinobacillus pleuropneumoniae

We report, for the first time, a chemiluminescence immunoassay (CLIA) method based on AuCl(4)(-)-enhanced luminol chemiluminescence (CL) reaction for the highly sensitive detection of ApxIV antibody of Actinobacillus pleuropneumoniae (APP). The AuCl(4)(-), which was the dissolution product of the gold nanoparticle-rabbit anti-pig IgG conjugate, served as an analyte in the CL reaction for the indirect measurement of antibody against ApxIV. The optimal condition of gold dissolution was composed of a 5.0 x 10(-2) M HCl, 1.5 x 10(-2) M NaCl, and 2.5 x 10(-4) M Br(2) solution. Under the optimal conditions, a good correlation between the relative CL photon counting and the dilution coefficient of serum was obtained in the dilution range of 1:160-1:40 000. Based on the analysis of clinical samples, the results indicated that CLIA had remarkable advantages in terms of reliability and practical use compared with indirect hemagglutination (IHA) and enzyme-linked immunosorbent assays (ELISA). The proposed method provided a new tool for the indirect determination of antibody against ApxIV in pig serum samples and showed great potential for numerous applications in immunoassays.     

化学发光酶联免疫分析测定血清中抗DNA抗体

建立起适于临床应用的抗ＤＮＡ抗体化学发光酶联免疫分析法。该法精密度良好，相对标准偏差为２．４％。比ＥＬＩＳＡ法更加简便、经济、省时，同时提高了灵敏度（８倍）和血清的阳性检出率。探讨了检测系统中对碘苯酚增强鲁米诺－过氧化氢－辣根过氧化物酶化学发光反应机理。     

Chemiluminescence detection coupled to high-performance frontal analysis for the determination of unbound concentrations of drugs in protein binding equilibrium

High-performance frontal analysis coupled with chemiluminescence detection (HPFA-CL) was developed for the determination of unbound oxacillin concentration in human serum albumin solution. The HPFA system consisted of an ISRP column and a mobile phase of 67 mM potassium phosphate buffer of pH 7.4 and ionic strength of 0.17. The luminol-H2O2-Co2+ system was used in the chemiluminescence detection. An enhancement of luminol chemiluminescence by oxacillin was investigated and employed for determining the concentration of oxacillin in the HPFA eluate. Sample solutions were directly injected onto the column; the drug was eluted as a zonal peak with a plateau region. The unbound drug concentrations were determined by using the height of the plateau. The results agreed with those obtained with conventional ultrafiltration-HPLC method. Good reproducibility was confirmed by the within run and between run RSD < or = 7.4%. HPFA-CL provided a selective method for determination of unbound drug concentration in protein binding equilibrium.     

Serum estradiol measurement by solid-phase chemiluminescence immunoassay and direct radioimmunoassay

Estradiol017β is determined in serum extracts by solid-phase chemiluminescence immunoassay. The results are compared with those obtained from unextracted serum in routine conditions with a commercial radioimmunoassay (r.i.a.) kit. For the chemiluminescence procedure, a purified monoclonal antibody to estradiol-6-carboxymethyloxime/bovine serum albumin and the homologous chemiluminescent marker conjugate estradiol-6-carboxymethyloxime aminobutylethylisoluminol are used. Bound and free ligand are separated by washing and simple centrifugation. Results obtained by the chemiluminescence assay (y) and by r.i.a. (x) on 170 serum specimens from women during ovulation induction showed good correlation (y = 1.01x ? 16 with r = 0.95). The methods are similar in selectivity, detection limit (ca. 10 ng l^?1) and precision (interassay relative standard deviation, 8–13%).     

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer–dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method. Principle of the U-MB real-time PCR method for HBV DNAdetection