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.     

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.     

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.     

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 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.     

Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay for Cellobiohydrolase I

A double-antibody sandwich enzyme-linked immunosorbent assay was developed for quantifying cellobiohydrolase I (CBH I) in crude preparations of the cellulase complex from Trichoderma reesei. The other enzymes (endoglucanase and β-glucosidase) in this complex and other ingredients in culture broth did not interfere with this assay. The antibody configuration that resulted in the highest specificity for the assay of CBH I employed a monoclonal antibody to coat wells in polystyrene plates and peroxidase-labeled polyclonal antibody to detect cellobiohydrolase bound to the immobilized monoclonal antibody. Previously, procedures have not been available for the direct assay of CBH I activity in the presence of the other enzymes in the complex, and current indirect procedures are cumbersome and inaccurate. The direct procedure described here is highly specific for CBH I and useful for quantifying this enzyme in the range of 0.1 to 0.8 μg/ml.     

Detection and Quantitation of Methanogens by Enzyme-Linked Immunosorbent Assay

Antisera to two methanogenic bacteria, Methanosarcina barkeri and a Methanobacterium sp., were raised in rabbits and used to develop an enzyme-linked immunosorbent assay (ELISA) method. ELISA was shown to be a sensitive technique, detecting as little as 4 ng of methanogen protein. The specificities of the antisera toward other methanogens were evaluated, and it was found that the antisera recognized species of the same genus as the immunizing species, but gave very little cross-reaction with methanogens of different genera. ELISA was used to estimate the growth of methanogens in pure culture. In natural environments and in anaerobic digesters methanogens exist as part of a mixed bacterial community, so the possibility of using ELISA to quantitate methanogens in mixed cultures was examined. The two antisera gave very little reaction in ELISA when non-methanogenic bacteria were used as antigens and ELISA was used to quantitate methanogens in an acetate enrichment culture. I conclude that the ELISA is a useful method for quantitating methanogens in defined mixed cultures, but has limited applicability to more complex systems.     

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.     

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.     

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。     

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     

Quantification of Dehalospirillum multivorans in Mixed-Culture Biofilms with an Enzyme-Linked Immunosorbent Assay

A fast, highly selective and sensitive method to quantify specific biomasses in mixed-culture biofilms is described. It consists of detachment of a biofilm from its support material, resolution of the detached biofilm flocs in order to separate the enclosed cells and antigens, and quantification of specific biomass by an enzyme-linked immunosorbent assay.     

Simplified Enzyme-Linked Immunosorbent Assay for Routine Identification of Rhizobium japonicum Antigens

A simple, reliable, and flexible modification of the indirect enzyme-linked immunosorbent assay was developed for the identification of Rhizobium japonicum antigens from cultures and nodules. The procedure emphasizes efficient use of time and reagents, adaptability to variously equipped laboratories, and maintenance of sensitivity levels that are adequate for ecological studies.     

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 of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

A Sandwich Enzyme-Linked Immunosorbent Assay for the Bacillus sphaericus Binary Toxin

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5α harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362. Received: 2 July 1997 / Accepted: 12 August 1997     

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。     

Indirect Enzyme-Linked Immunosorbent Assay Based on the Nucleocapsid Protein of SARS-like Coronavlruses

The nucleocapsid protein （N） is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus （SL-CoV） has a high similarity with that of SARS-CoV. In this study, the SL-CoV N protein was expressed in Escherichia coli, purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay （indirect ELISA） was developed for detection of SARS- or SL-CoV infections in bat populations. The detection of 573 bat sera with this indirect ELISA demonstrated that SL-CoVs consistently circulate in Rhinilophus species, further supporting the proposal that bats are natural reservoirs of SL-CoVs. This method uses 1-2 μl of serum sample and can be used for preliminary screening of infections by SARS- or SL-CoV with a small amount of serum sample.