对氟苯酚—H2O2—HRP体系酶联荧光免疫分析研究：IV,F^—Zr—8羟基喹…

将Ｚｒ－８羟基喹啉－５－磺酸荧光体系与对氟苯酚为底物的酶促反应相偶合，建立了一种测定辣根过氧化物酶（ＨＲＰ）及其标记物的新方法，测定ＨＲＰ的线性范围为０．０３１～３１ｍＵ／ｍＬ，检出限（３σ）为０．００７ｍＵ／ｍＬ．用于测定人血清中乙肝表面抗原和Ｅ抗体。结果令人满意.     

以氟苯酚—H2O2—HRP体系酶联荧光免疫分析研究：Ⅲ.F^—Al酸性铬蓝…

建立了一种检测人血清中乙肝Ｅ抗原的新荧光光度法，通过酶促反应，对氟苯酚＋Ｈ２Ｏ２→＾ＨＲＰ苯酚＋Ｆ＾－＋Ｈ２Ｏ与Ａｌ－酸性铬蓝Ｋ荧光体系相偶合，测定辣根过氧化物酶（ＨＲＰ）及其标记物，测定ＨＲＰ的线性范围为０．１９～３１ｍＵ／ｍＬ，检出限为０．０４ｍＵ／ｍＬ。     

对氟苯酚-H_2O_2-HRP体系酶联荧光免疫分析研究ⅢF~--Al-酸性铬蓝K荧光熄灭法测定人血清中乙肝E抗原

建立了一种检测人血清中乙肝Ｅ抗原的新荧光光度法．通过酶促反应：对氟苯酚＋Ｈ２Ｏ２ＨＲＰ→苯酚＋Ｆ－＋Ｈ２Ｏ与Ａｌ－酸性铬蓝Ｋ荧光体系相偶合，测定辣根过氧化物酶（ＨＲＰ）及其标记物．测定ＨＲＰ的线性范围为０．１９～３１ｍＵ／ｍＬ，检出限为０．０４ｍＵ／ｍＬ．     

对氟苯酚-过氧化氢-辣根过氧化物酶体系酶联荧光免疫分析研究──Ⅰ.对氟苯酚直接法及铝-钙指示剂偶合法测定人血清中乙肝表面抗原和表面抗体

对氟苯酚在辣根过氧化物酶（HRP）的催化下可被H2O2氧化，生成本酚及F-。测定对氟苯酚荧光猝灭程度及用铝－钙指示剂荧光体系检测酶促反应释放的F－，可分别建立HRP及其标记物的两种分析方法。铝－钙指示剂偶合法测定HRP的线性范围为0．031～31U／L，检测限（3δ）为0．0093U／L。两种方法分别用于测定人血清中乙肝表面抗原及抗体，结果令人满意。     

对氟苯酚-H_2O_2-HRP体系酶联荧光免疫分析研究ⅣF~--Zr-8-羟基喹啉-5-磺酸荧光法测定人血清中乙肝表面抗原和E抗体

将Ｚｒ－８－羟基喹啉－５－磺酸荧光体系与对氟苯酚为底物的酶促反应相偶合，建立了一种测定辣根过氧化物酶（ＨＲＰ）及其标记物的新方法．测定ＨＲＰ的线性范围为０．０３１～３１ｍＵ／ｍＬ，检出限（３σ）为０．００７ｍＵ／ｍＬ．用于测定人血清中乙肝表面抗原和Ｅ抗体，结果令人满意．     

OT-H2O2-HRP伏安酶联免疫分析新体系

本文首次提出了邻联甲苯胺(OT)-H2O2-辣根过氧化物酶(HRP)伏安酶联免疫分析新体系。本方法以线性扫描二阶导数伏安法检测HRP催化H2O2氧化OT的产物, 用于游离HRP和各种HRP标记物测定, 灵敏度比经典的ELISA光度法分别高两个至四个数量级。测定游离HRP的检测限达到1.8×10^-^1^2 g/mL, 线性范围为5.0×10^-^1^2-1.0×10^-^8 g/mL。对此伏安酶联免疫分析新体系的偶合反应机理及电极还原过程也进行了详细的研究。     

甲基红-H2O2-HRP伏安酶联免疫分析法测定HRP及其标记物

提出了一种新的辣根过氧化物酶的底物-甲基红,它本身具有电化学活性,能够在静汞电极上发生还原反应,产生灵敏的伏安电流信号.以H2O2为氧化剂,HRP能催化氧化还原反应的发生,使甲基红被氧化分解,其平衡浓度降低,对应的还原峰电流降低,峰电流的降低值与HRP的质量浓度在5.0×10-8～5.0×10-7g/mL之间呈线性关系,对2.0×10-7g/mL HRP进行11次测定的相对标准偏差为4.6%,方法的检出限为1.8×10-8g/mL.应用于IgG-HRP和Avidin-HRP的测定.     

联苯胺—H2O2—HRP偶合反应伏安酶联免疫分析新体系测定HRP的研究

提出了联苯胺－Ｈ２Ｏ３－ＨＲＰ偶合反应伏安酶联免疫分析新体系测定ＨＲＰ的方法。通过测定ＨＲＰ催化Ｈ２Ｏ２氧化联苯胺的产物间接测定ＨＲＰ的浓度。     

酸性铬蓝K-H2O2-HRP伏安酶联免疫分析法测定HRP及其标记物

本文提出了一种新的辣根过氧化物酶(HRP)的底物--酸性铬蓝K(ACBK),它本身具有电化学活性,能够在汞电极上发生还原反应.以H2O2为氧化剂,HRP的加入能加快氧化反应的进行,使酸性铬蓝K被氧化分解,其平衡浓度降低,对应的还原峰电流降低,峰电流的降低值同HRP的加入量在8.0×10-8～1.0×10-6 g/mL之间呈线性关系.用于IgG-HRP的测定,最高稀释比为1∶5 000.     

Gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis B surface antigen in human serum

The application of gold nanoparticle-based electrochemical immunoassays have been extensively studied for the detection of hepatitis B surface antigen (HBsAg), but most often they exhibit low sensitivity. We describe the fabrication of a new electrochemical immunoassay for signal amplification of the antigen-antibody reaction combined with the nanogold-based bio-barcode technique. Hepatitis B surface antibody (HBsAb) was initially immobilized on a nanogold/thionine/DNA-modified gold electrode, and then a sandwich-type immunoassay format was employed for the detection of HBsAg using nanogold-codified horseradish peroxidase-HBsAb conjugates as secondary antibodies. Under optimal conditions, the current response of the sandwich-type immunocomplex relative to the H₂O₂ system was proportional to HBsAg concentration in the range from 0.5 to 650 ng·mL^?1 with a detection limit of 0.1 ng·mL^?1 (S/N?=?3). The precision, reproducibility and stability of the immunosensor were acceptable. Subsequently, the immunosensors were used to assay HBsAg in human serum specimens. Analytical results were in agreement with those obtained by the standard chemiluminescence enzyme-linked immunosorbent assay.     

Expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system

The expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system was investigated. The human TK- 143 cells were infected with the recombinant vaccinia viruses vTMS-1 or vTLS-1. Cells infected with vTMS-1, which contains the preS2 + S gene, produced preS2 containing middle HBsAg proteins. Similarly, cells produced preS1 containing large HBsAg proteins upon infection with vTLS-1, which carries the preS1 + preS2 + S gene. The expression products could be secreted and form 22 nm particles. They reacted specifically with anti-preS1 and/or anti-preS2 monoclonal antibodies, and exhibited pHSA-receptor (for polymerized human serum albumin) activity. In addition, the major S components of hepatitis B surface antigen were also present in the products expressed by vTMS-1 and vTLS-1.     

Ultra-sensitive immunosensor for detection of hepatitis B surface antigen using multi-functionalized gold nanoparticles

The signal amplification for analytical purposes has considerable potential in detecting trace levels of analytes for clinical, security or environmental applications. In the present report a strategy based on a sandwich type immunoassay system was designed for the detection of hepatitis B surface antigen which exploits the specific affinity interaction between streptavidin and biotin recognition systems. The method involves the specific coupling of multi-functionalized gold nanoparticles (bearing biotin and luminol molecules) to the streptavidin modified by secondary antibody. The chemiluminescent signal is produced by the gold nanoparticles in the presence of HAuCl₄ as catalyst and hydrogen peroxide as oxidant. The immunosensor was able to detect hepatitis B surface antigen in the linear concentration range from 1.7 to 1920 pg mL⁻¹ and the detection limit of 0.358 pg mL⁻¹, at signal/noise = 3.     

Size exclusion chromatography of hepatitis B surface antigen particles

Summary To find the factors responsible for the broadening of the recombinant-hepatitis B surface-antigen peak in size-exclusion chromatography, the purified material was fractionated on preparative scale followed by multiple analysis of the separated fractions. The results from chromatographic analysis suggested the presence of large particle aggregates, probably tubular structures which, however, were not detected by electron microscopy. The antigen particles ranged from 16 to 32 nm in all the fractions, except two last fractions consisting of 16–24 nm particles. The relation ELISA/Lowry increased with increasing the fraction number, being a maximum in the fraction corresponding to the maximum of the chromatographic peak. Probably, the particles which are variable in size differ from each other with respect to the efficiency of protein assembly. Fractions collected in different regions of the peak were adsorbed on alum and injected in mice. The high antibody levels were produced without significant differences in immunogenicity between samples. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

A fluoroimmunoassay for screeing of hepatitis B surface antigen

A fast, sensitive sandwich fluorimmunoassay for hepatitis B surface antigen (HB_s-ag) is described. Rabbit antibodies to HB_s-ag are covalently bound to the centre of a glass-fiber filter disk. The second antibody is a swine anti-human HB_s-ag conjugated with β-d-galactosidase from E. coli. The substrate is 4-methylumbelliferyl-β-d-galactopyrano-side. The technique is applied with the Stratus system which does the entire test in 8 min. the detection limit is 1 ng ml^?1, and the results correlate well with a radioimmunoassay technique.     

Preparation and characterization of biodegradable microspheres containing hepatitis B surface antigen

Poly-DL-lactide-poly(ethylene glycol) (PELA) microspheres containing Hepatitis B surface antigen (HBsAg) were elaborated by a solvent extraction method based on the formation of a double water/oil/water (w/o/w) emulsion. Microspheres were characterized in terms of morphology, size and size distribution, encapsulation efficiency, and the efficiency of microsphere formation (EMF). Transmission electron microscopy (TEM) and polyacrylamide gel electrophoresis (PAGE) were used to investigate the structural integrality of HBsAg encapsulated in PELA microspheres. The release profile was investigated by the measurement of antigen present in the release medium at various intervals. The PELA-10 microspheres displayed the highest antigen encapsulation efficiency (about 80%), and antigen molecules could be stabilized in the PELA-10 microspheres during the preparation process. It suggested that the PELA microspheres had a great potential as a new polymer adjuvant for HBsAg. The release of Hepatitis B surface antigen from poly-DL-lactide-poly(ethylene glycol) microspheres.     

Determination of catecholamine in human serum by a fluorescent quenching method based on a water-soluble fluorescent conjugated polymer-enzyme hybrid system

In this paper, a sensitive water-soluble fluorescent conjugated polymer biosensor for catecholamine (dopamine DA, adrenaline AD and norepinephrine NE) was developed. In the presence of horse radish peroxidase (HRP) and H(2)O(2), catecholamine could be oxidized and the oxidation product of catecholamine could quench the photoluminescence (PL) intensity of poly(2,5-bis(3-sulfonatopropoxy)-1,4-phenylethynylenealt-1,4-poly(phenylene ethynylene)) (PPESO(3)). The quenching PL intensity of PPESO(3) (I(0)/I) was proportional to the concentration of DA, AD and NE in the concentration ranges of 5.0 × 10(-7) to 1.4 × 10(-4), 5.0 × 10(-6) to 5.0 × 10(-4), and 5.0 × 10(-6) to 5.0 × 10(-4) mol L(-1), respectively. The detection limit for DA, AD and NE was 1.4 × 10(-7) mol L(-1), 1.0 × 10(-6) and 1.0 × 10(-6) mol L(-1), respectively. The PPESO(3)-enzyme hybrid system based on the fluorescence quenching method was successfully applied for the determination of catecholamine in human serum samples with good accuracy and satisfactory recovery. The results were in good agreement with those provided by the HPLC-MS method.