用酶联免疫吸附法检测蛋白质的聚合

许多蛋白质二聚化或多聚化在调节其功能方面发挥重要作用,研究蛋白质的聚合有助于阐明相关的生物学过程. 本文使用酶联免疫吸附法,对分子量11 kD的马传染性贫血病毒核壳蛋白（nucleocapsid protein 11 kD, NCp11）的聚合进行检测. 首先利用亲和层析分别纯化了NCp11以及N 端或C 端融合有FLAG标签的NCp11. 然后将NCp11包被于聚苯乙烯96孔板底,加入带FLAG标签的NCp11与之聚合,再依次加入抗FLAG抗体、辣根过氧化物酶标记的二抗及底物,反应终止后于450 nm波长下读取吸收值. 结果表明,酶联免疫吸附法适用于NCp11聚合的检测,可对聚合的特异性、剂量依赖效应和影响因素等进行定量评价. 利用该方法不仅能检测蛋白质的聚合,而且具有灵敏度高、特异性好、通量高、成本低和快速简便等优点,有望获得广泛应用.     

Enzyme-Linked Immunosorbent Assay for Detection of Chitooligosaccharides

In order to detect chitooligosaccharides (COS), an enzyme-linked immunosorbent assay (ELISA) was developed. A chitooligosaccharide mixture (COSM) conjugated to bovine serum albumin was used to immunize rabbits to produce an anti-COS polyclonal antibody. By use of specific antibody and COSM-horseradish peroxidase conjugate, we established a competitive direct ELISA (cdELISA) the detection limit of which was about 0.1 μg/ml. In the cdELISA, the cross-reactivities of the specific antibody toward glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose were 0.27, 27, 75, 75, 144, and 100%, respectively, and those toward N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose were 1.58, 0.005, 1.08, 0.05, and 0.40%, respectively.     

Rubisco Quantitation in Leaves of Different Barley Varieties by Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) for the quantitationof Rubisco was developed. The amount of Rubisco in the primaryleaves often different barley varieties was determined. It variedfrom 15 to 50 per cent of the total soluble protein and from2·5 to 10 mg g^–1 fresh weight. The carboxylaseand oxygenase activities of the extracts were consistent withthe results of the immunoenzymatic assay. The variability ofthe Rubisco content among varieties suggests genetic determination. Key words: Rubisco, ELISA, Hordeum vulgare     

Enzyme-Linked Immunosorbent Assay for Detection of Ochratoxin A

A simple microtest plate enzyme-linked immunosorbent assay was developed for the detection of ochratoxin A at levels as low as 25 pg per assay. The relative cross-reactivities of the antibody in this system with ochratoxin A (OA), OB, OC, and Oα were found to be 1.0, 0.14, 0.44, and 0.01, respectively.     

单克隆抗体在淋病奈瑟氏菌研究和感染诊断中的应用

淋病是全球性疾病,对于淋球菌的研究已进入分子水平,本文从以下３个方面综述了单克隆体抗体在淋球菌研究和感染诊断中的应用：１．对于脂寡糖的研究２．对于淋球菌外膜蛋白Ⅰ的研究３．单克隆抗体在淋病流行病学和诊断中的应用。     

酶联免疫吸附试验检测A型产气荚膜梭菌肠毒素

本实验建立了双抗体夹心ＥＬＩＳＡ检测产气荚膜梭菌肠毒素的实验体系。以家兔单特异ＩｇＧ包被酶标板,辣根过氧化物酶标记的绵羊ＩｇＧ作为指示物,可检测出１．２５ｎｇ／ｍｌ（０．１２５ｎｇ）的产气荚膜梭菌肠毒素,线性范围大,重复性及稳定性好,对培养上清及粪便滤液检查无非特异反应。是产气荚膜梭菌食物中毒实验室诊断的一种快速可靠的检测方法。     

Enzyme-Linked Immunosorbent Assay for Quantitative Detection of Bacillus thuringiensis Crystal Protein

Accurate measurement of the toxic protein crystal produced during deep-tank fermentation of Bacillus thuringiensis is critical for optimum process yield. The currently accepted method is a bioassay that requires more time to generate data than to complete the fermentation itself. A noncompetitive enzyme-linked immunosorbent assay has been developed with purified B. thuringiensis crystals to generate rabbit antiserum. This technique gives a quantitative crystal protein value with a colorimetric endpoint for either liquids or powders within 4 h of sampling. Reproducibility of this enzyme-linked immunosorbent assay satisfies criteria for use in a commercial process.     

酶联免疫吸附测定法

酶联免疫吸附测定法（ELISA）是一种灵敏度、特异性和精度都十分优良的测定方法。在诊断、疾病监控以及研究用途方面得到了广泛的运用。现在还在对其进行开发，以简化工序，提高灵敏度。此讲由SRL公司试药部的原田弘智对其进行讲解。[编者按]     

Enzyme-Linked Immunosorbent Assay of Fungal NADP-Glutamate Dehydrogenase

A sensitive and reliable method has been developed for the quantitation of NADP⁺-glutamate dehydrogenase from the phytopathogenic Ascomycete Sphaerostilbe repens using a two-step competitive enzyme-linked immunosorbent assay. Purified enzyme was adsorbed noncovalently to polystyrene wells and rabbit immunserum was allowed to bind to antigensensitized wells. Bound specific antibody was visualized by goat antirabbit immunoglobulin covalently linked to alkaline phosphatase using paranitrophenylphosphate as the substrate. Increasing amounts of purified enzyme or crude fungal extracts were quantitated by their ability to inhibit specific antibody adsorption to antigen-coated polystyrene wells. This system proves to be useful in the range of 10 to 80 nanograms of enzyme level. Using this assay, identical amounts of NADP⁺-glutamate dehydrogenase were found in mycelia grown on nitrate and ammonia sources.     

酶联免疫吸附检测法的应用研究进展

酶联免疫吸附检测法(ELISA)由于具有灵敏、特异、简单、快速及易于自动化操作等优点而得到了快速发展,并且被广泛应用于多个领域.本文对ELISA检测法应用现状及优缺点进行综述,为今后ELISA检测法的更为广阔的应用与发展提供参考.     

Detection of Four Seed-borne Plant Viruses by the Enzyme-Linked Immunosorbent Assay (ELISA)

This study was conducted to evaluate the sensitivity of the ELISA technique in detecting four economically important viruses, namely barley stripe mosaic (BSMV), cucumber green mottle mosaic (CGMMV), bean common mosaic (BCMV), and squash mosaic (BSMV) viruses in single seeds as well as in batches of barley, cucumber, bean and squash seeds, respectively. Results indicated the suitability of the technique in detecting the above viruses in single germinated seeds or embryos. Accordingly, seed transmission rates of BSMV, CGMMV, BCMV and SqMV were found to be 67 %, 17%, 17% and 12%, respectively. In artificially contrived mixtures of infected: healthy seeds or embryos, BSMV, CGMMV, BCMV and SqMV were successfully detected at ratios of 1 : 500, 1 : 25, 1 : 10 and 1 : 10, respectively. Sensitivity of detection was increased in the ease of BSMV by using germinated rather than ground dry BSMV-infected barly seeds; and in the case of SqMV, by using whole germinating emybryos rather than coleoptiles only. Trials on re-using the enzyme-γ-globulin conjugate indicated that CGMMV conjugate used once can be re-used with little loss in reactivity.     

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

A 1-Hour Enzyme-Linked Immunosorbent Assay for Quantitation of Acrolein- and Hydroxynonenal-Modified Proteins by Epitope-Bound Casein Matrix Method

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibody specific for acrolein-modified lysine or HNE-modified histidine epitopes. Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules.     

基于磁性纳米微球的Gamma干扰素酶联免疫检测方法建立

目的：建立及评价使用磁性纳米微球作为固相载体的人酌干扰素（Interferon-gamma,IFN-gamma）双抗体夹心酶联免疫吸附实验 （Enzyme-linked immunosorbent assay,ELISA）检测方法。方法：以杂化细乳液合成法制备磁性纳米微球,将其作为免疫检测的固相 载体。将磁性微球与IFN-酌抗体进行偶联,建立基于磁性微球的ELISA 检测方法,检测人IFN-gamma,绘制IFN-gamma标准曲线并进行方法 学评价。结果：获得包被有人IFN-gamma抗体的免疫微球, 抗体偶联率为54.5 %。用它建立IFN-gamma的双抗体夹心的ELISA 检测方法,检 测范围为0-1000 pg/mL,相关系数为0.9996,灵敏度23.2 pg/mL,功能灵敏度0 pg/mL,批内和批间变异系数（Coefficients of Variance,CVs）＜8 %,检测总共需要2 小时。结论：成功制备了IFN-酌免疫微球并建立了定量检测人IFN-gamma的双抗体夹心磁珠 酶联免疫方法。     

Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.     

Serological Diagnosis of Human Babesiosis by IgG Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies to Babesia microti antigen was developed. B. microti antigens were harvested from experimentally infected hamster blood and used as a coating antigen. The sensitivity and specificity of the IgG ELISA relative to immunofluorescent antibody assay (IFA) testing was 95.5% and 94.1%, respectively. According to the receiver operating characteristic curve analysis, the area under the curve was 0.987. No cross-reactivity of serum samples collected from patients infected with Toxoplasma gondii, Borrelia burgdorferi, Anaplasma phagocytophilum, Bartonella quintana, Dengue virus, or West Nile virus was detected. Cross-reactivity was observed with one of 35 sera from patients infected with Bartonella henselae. These results indicate that the established ELISA methods could be utilized as an accurate measure for the clinical diagnosis of human babesiosis.     

Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Pseudomonas fluorescens Proteases in Ultrahigh-Temperature-Treated Milk

An inhibition enzyme-linked immunosorbent assay was developed to detect low levels of the proteases extracted from four strains of Pseudomonas fluorescens. The assay detected between 0.24 and 7.8 ng of protease per ml of ultrahigh-temperature-treated milk and could be completed within 6 h. It could be used as a framework for a test system for quantifying spoilage proteases in dairy products.