荧光酶标免疫分析在食品微生物检测中的应用

荧光酶标免疫分析是一种新型的快速检测微生物的方法,具有方便、快速、灵敏、准确等优点,已越来越多地被用于进出口食品中致病菌的检测。本文介绍了荧光酶标免疫分析法的原理、特点以及在食品微生物检测中的应用和未来发展方向。     

磁性均相酶联免疫分析系统高技术产业化

该项目采用自主开发的纳米磁性微珠生产技术,创造性地将纳米磁性微珠分离技术和酶联免疫分析技术相结合,用碱性磷酸酶代替经典酶联免疫分析的辣根过氧化物酶，建立了一种全新的酶联免疫分析方法，使得该项目生产的磁酶免疫试剂、测定分析仪等系列产品在分析的灵敏度、准确率、精密度、特异性等方面拥有传统的放射免疫分析和酶联免疫分析所无法达到的性能优势，技术水平国内领先。[第一段]     

时间分辨荧光免疫分析及其在临床检测中的应用

本文介绍了时间分辨荧光免疫分析法的检测原理、检测方法,分析了时间分辨荧光免疫分析仪的结构并介绍了其在临床检测方面的应用。     

免疫标记技术的进展及纳米技术在其中的应用

酶免疫标记、胶体金免疫标记及荧光免疫标记技术是目前应用最广泛的免疫反应检测技术。随着科学技术,尤其是纳米技术的发展和生物医药等方面的需求,人们在提高检测灵敏度、简化操作步骤、有效缩短检测时间等方面,取得了很多可喜的进展。本文将分别针对酶免疫标记、胶体金免疫标记及荧光免疫标记技术三方面的进展情况,尤其是纳米标记技术在免疫反应检测中的应用进展进行综述。     

时间分辨荧光免疫分析仪的临床实验方法和评价方案

时间分辨荧光免疫分析技术是一种利用稀土离子及其螯合物作为示踪剂的灵敏度高、线性范围宽、应用范围广的非放射性标记免疫分析技术。研制了一种性能稳定的时间分辨荧光免疫分析仪,为了进一步将这一先进超微量物质检验技术推广于临床应用,根据美国国家临床实验室标准化委员会（NCCLS）制订的临床评价方案,选择放射免疫分析法、化学发光免疫分析法以及Perkin Eimer Life Sciences公司的Auto DFLFIA-1235全自动时间分辨荧光免疫分析仪作为比较方法,提出了三套检验时间分辨荧光免疫分析仪性能的临床实验方法和评价方案。并利用其中的一套方案进行了实验,结果表明这些方案可操作性强,结果可信,经济适用,可作为同类医疗检验仪器进行临床实验的参考。     

免疫法在食品分析中的应用

免疫法在食品分析中的应用卢大用龚镥（上海大学生命科学学院,上海２０１８００）由于当今的食品分析已包容了物理,化学,生物及微生物等诸多手段,在不同类型方法的配合及竞争中,免疫分析法可谓发展迟,后劲足,如农药等的检测中,很多美国的科技人员及企业已正式提出...     

压电免疫传感器固定方法

抗原抗体在石英晶体电极表面固定而不丧失其活性是压电免疫传感器成功的关键,直接影响到它的灵敏度和可重复性等性质。介绍了压电免疫传感器传统的表面固定方法:主要有戊二醛交联法、自组装单分子膜法(SAM)、蛋白A固定法;以及国内外最新研究的不需通常固定化步骤的方法,主要是压电凝胶免疫分析法(LEPIA)和PEG压电凝胶分析法,并对这些方法的发展前景做了展望 。     

流感病毒血凝素基因、神经氨酸酶基因在小鼠中的免疫效果观察

流感病毒血凝素基因,神经氨酸酶基因免疫BALB／c小鼠,获得特异阳性抗体反应,抗体滴度与基因免疫量呈正相关性,各实验组免疫小鼠抗同型流感病毒攻击存活率为100％,血凝素基因免疫小鼠抗异型流感病毒攻击存活率为100％,神经氨酸酶基因免疫小鼠抗异型流感病毒攻击存活率为75％,血凝素基因与神经氨酸酶基因联合免疫小鼠抗异型流感病毒攻击存活率为100％。     

低水平乙型肝炎表面抗原的检测及其临床价值评估

我们采用具有世界卫生组织( WHO) 溯源性的标准血清建立浓度标准曲线, 评估微粒子酶免疫分析法(MEIA) 和酶联免疫吸附试验( ELISA) 的检测灵敏度, 并对97 例经确认为低水平乙型肝炎表面抗原( HBsAg)的阳性结果进行检测和比对分析; 同时综合评估低水平HBsAg 患者的临床各项指标, 探究低水平HBsAg 检测的临床意义。根据MEIA、ELISA 的S/CO 值分析, 2 种方法的检测灵敏度分别为0. 095 IU/ml 和0. 378 IU/ml;ELISA 对97 例低水平HBsAg 的检出率仅为45. 4% ( 44 /97) ; 在97 例低水平HBsAg 患者中, HBV DNA≥1 ×103 拷贝/ml 占17. 5% ( 17/97) , 肝功能异常率占38. 1% ( 37 /97) , 其中明确诊断为乙型肝炎相关疾病的19 例患者中, 肝硬化占57. 9% ( 11 /19) 。因此灵敏度高、特异性好的方法有助于低水平HBsAg 的检出, 有利于对这些人群的诊断和治疗。部分低水平HBsAg 患者往往伴随病毒复制及肝功能的慢性损伤, 预后不良, 应引起临床的高度重视和关注。     

免疫亲和层析法纯化苦瓜几丁酶

用扁豆几丁酶免疫家兔,获得抗扁豆几丁酶的抗体,将此抗体与Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联,制备免疫亲和吸附剂,用以纯化苦瓜几丁酶．苦瓜叶片的粗提液经过免疫亲和吸附柱后,可获得电泳纯的几丁酶,其分子量为３５ ｋＤ,与用几丁质凝胶为亲和吸附剂的纯化结果一致．表明利用植物几丁酶在结构上的保守性,用免疫亲和法可纯化不同植物的同类几丁酶．与几丁质凝胶亲和柱相比,免疫亲和法纯化植物几丁酶具有快速、亲和柱可重复使用等的优点．利用免疫亲和层析获得的纯化样品,研究了苦瓜几丁酶对真菌的抑制试验,研究结果表明,苦瓜几丁酶能分解棉花枯萎病菌的菌丝体细胞壁制备物,并对其孢子芽管的伸长有一定抑制作用．     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

An enzyme immunoassay for plasma betamethasone

A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-β-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-β-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using ³H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol was 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.     

Assessment of mucosal immune response in genitourinary tract using urine.

A novel method to assess mucosal immune response in the genitourinary mucosa after immunization with a mucosal vaccine has been developed. In this method, secretory IgA antibody is measured by a highly sensitive enzyme immunoassay (immune-complex transfer enzyme immunoassay) using urine as a specimen. The urinary IgA antibody response could be detected by the immune-complex transfer enzyme immunoassay. In contrast, a conventional enzyme immunoassay (enzyme-linked immunosorbent assay (ELISA)) could not detect this response because of its low sensitivity. Because urine samples can be collected easily and nontraumatically, not only from experimental animals but also from humans, both males and females, the present method may be applicable for assessing the protective efficacy of candidates for mucosal vaccines against sexually transmitted microorganisms, such as human immunodeficiency virus. Furthermore, the usefulness of this method for novel mucosal vaccine formulae was shown for a model in which vaccine antigen and Bordetella pertussis adjuvant were adsorbed onto CaCO, and enclosed in enteric coated capsules.     

An enzyme immunoassay of estrone in swine serum

An enzyme immunoassay for estrone in swine serum was established. For this, beta-galactosidase from E. coli was conjugated through estrone-17 (O-carboxymethyl)oxime using a mixed anhydride reaction. The percentage of immunoreactive estrone-17 (O-carboxymethyl)oxime-beta-galactosidase conjugate was estimated to be about 70%. The recovery rate of estrone (25-500 pg) added to 0.05 ml of swine serum averaged 91.4%. The sensitivity of the present enzyme immunoassay was 5 pg/tube. The coefficients of variation (CV) were 5.9-8.2% (within assays) and 4.1-5.9% (between assays), respectively. Estrone values determined by the present enzyme immunoassay were highly correlated with those determined by radioimmunoassay (r = 0.99, P less than 0.005). This method of enzyme immunoassay was determined to be suitable for the routine assay of serum estrone.     

Enzyme immunoassay for cortisol in serum using cortisol 21-amine

Cortisol 21-amine (21-amino-11β,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 μg/d1, using 10 μ1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme Immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.     

A sensitive bridge heterologous enzyme immunoassay of progesterone using geometrical isomers

A sensitive bridge heterologous enzyme immunoassay of progesterone using geometrical isomers of progesterone 3(O-carboxymethyl)oxime(E/Z) was developed. Isomers were separated by synthesis of N-hydroxysuccinimide esters. Progesterone 3(Z)(O-carboxymethyl)oxime N-hydroxysuccinimide ester bound with beta-galactosidase in an appropriate molar ratio provided a conjugate suitable for an enzyme immunoassay. The antiserum was raised in rabbits by immunizing the animals with the progesterone 3(E)(O-carboxymethyl)oxime-bovine serum albumin conjugate. This bridge heterologous enzyme immunoassay proved to have sufficient sensitivity equivalent to radioimmunoassay and excellent specificity.     

An enzyme immunoassay for secretin

A sensitive and specific enzyme immunoassay for secretin was developed with the use of enzyme-labeled antigens. Synthetic porcine secretin and its carboxy-terminal fragments (residues 11-27 and 18-27) were conjugated with beta-D-galactosidase for use in the immunoassay, and the assay method with the latter fragment (residues 18-27) linked to beta-D-galactosidase was found to be the most sensitive. The minimum amount of secretin detectable by this method was 1-2.5 pg/assay. Serum levels of secretin after intravenous injection of the peptide in rats were determined by both the enzyme immunoassay and a commercial radioimmunoassay kit. The correlation coefficient between the levels measured by the two methods was 0.984. The enzyme immunoassay could detect immunoreactive secretin levels in normal human sera, giving a value of 16.9 +/- 2.2 pg/ml (mean +/- SE of six human subjects).     

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.     

A new sensitive and fast peptide immunoassay based on enzyme amplification used in the determination of CGRP and the demonstration of its presence in the thyroid

An enzyme amplified immunoassay for rCGRP based on cofactor cycling has been found to be clearly superior to a comparable radioimmunoassay employing the same antiserum in terms of sensitivity, speed and convenience. Correlation between the two methods was very good. With the enzyme amplified immunoassay we have been able to demonstrate the existence of rCGRP in thyroid extract.     

Comparison of quantity of thyroxine in thyroglobulin and the peptide hormones derived from chromatography and immunoenzyme assay

An evaluation of the thyroxine content in thyroglobulin and different derived thyroxine-containing peptides by enzyme immunoassay is presented. Results correlate very well with those obtained by ion-exchange chromatography which served as a reference method: r = 0.997, p less than 10(-6). Enzyme immunoassay is more rapid and sensitive than ion-exchange chromatography and then suitable for routine use. Furthermore, this paper shows, for the first time, that enzyme immunoassay can be used to perform direct thyroxine estimation in small peptide (Mr less than 2,500) without previous total enzymatic hydrolysis.