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.     

Assessment of Red Core Resistance of Strawberry Using Enzyme-Linked Immunosorbent Assay (ELISA)

There was a strong correlation between measurements of red core disease of strawberry, assessed as percentage lengths of the adventitious roots with red steles, and ELISA values obtained from the same root systems using a polyclonal antiserum raised to Phytophthora fragariae. ELISA grouped twelve host cultivar/pathogen isolate combinations into three categories, highly resistant, partially resistant and susceptible, which agreed with disease incidence after inoculation.     

Enzyme-Linked Immunosorbent Assay (ELISA) of a Coryneform Isolated from Ratoon Stunted Sugarcane

An enzyme-linked immunosorbent assay (ELISA) was developed for the coryneform bacterium isolated from ratoon stunted surgarcane. Parameters governing the ELISA were optimized. These included antibody coating concentration, conjugate concentration and diluent, antigen diluent, the washing procedure, incubation temperature for sample and conjugate, pH and time. The level of detectability of the cultured bacterium by the ELISA was ≥ 1 × 10⁴ Zellen · ml^-1.     

The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

Background

Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis.

Methodology/Principal findings

Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases.

Conclusions/Significance

A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.     

High Seroprevalence of Mycoplasma pneumoniae IgM in Acute Q Fever by Enzyme-Linked Immunosorbent Assay (ELISA)

Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA), a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001) and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001) of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5%) and seroconversion rate (33.3%) of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases) with those who were negative (43 cases), the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255), sore throat (8.5% vs. 16.3%, p=0.351), cough (35.6% vs. 23.3%, p=0.199), and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258), were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.     

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.     

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.     

Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots

The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 10⁵ CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 10⁸ CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.     

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.     

The Use of Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Cacao Swollen Shoot Virus (CSSV) in Theobroma cacao

Cacao swollen shoot virus (CSSV) was readily detected in different parts of Theobroma cacao using the ELISA technique. Different plant tissues contained varying amounts of CSSV; highest concentrations were found in leaf lamina. Methods to preserve the serological activity of CSSV were evaluated, and best results obtained with samples stored in a buffer or freeze-dried.     

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.     

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。     

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。     

Identification of Heat-Stable Enterotoxin-Producing Strains of Yersinia enterocolitica and Vibrio cholerae Non-O1 by a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay

Using a mouse monoclonal antibody (MAb) 2F raised against Vibrio cholerae non-O1 heat-stable enterotoxin (NAG-ST) which also recognizes a shared epitope of Yersinia enterocolitica heat-stable enterotoxin (Y-ST), a competitive enzyme-linked immunosorbent assay (ELISA) was developed for independent detection of NAG-ST and Y-ST. There was good concordance between the Y-ST ELISA and the suckling mouse assay (SMA) for detection of Y-ST from test strains of Y. enterocolitica, and the Y-ST ELISA can effectively replace the SMA for routine detection of Y-ST. On the contrary, evaluation of the NAG-ST ELISA and the SMA using 139 strains of V. cholerae non-O1 showed discordant results and this was attributed to the presence of the suckling mice active factor(s) such as El Tor hemolysin and to the production of low amounts of NAG-ST. Concentration of culture supernatants of V. cholerae non-O1 followed by heating at 100 C was essential to obtain reproducible results by both the NAG-ST ELISA and the SMA. The ELISA developed in this study can be used for the identification of biologically active strains. While recently genetic methods such as polymerase chain reaction became available and were very reliable and simple techniques, the ELISA in this study has an advantage in detecting biologically toxic gene products of the strains. The genetic methods cannot differentiate silent STa genes which we often encounter in the case of Y. enterocolitica.     

Enzyme-Complemented Activatorsorbent Assay (ECASA): Genetic Engineering for Enzyme-Linked Immunosorbent Assay-Type Mercuric Ion Detection

The sensor component of bacterial mercury resistance systems is the metalloregulatory protein MerR, which has nanomolar sensitivity and high selectivity for Hg(II). A fusion protein of MerR and the α-peptide part of β-galactosidase (LacZα) was constructed by fusing the relevant genes. The protein exhibited both MerR functions and α-complementing activity to the inactive LacZΔM15 (M15) protein. The bifunctional character of the appropriate MerR–LacZα-complemented M15 protein (MerR–LacZα:M15 protein complex) was used to develop a Hg(II)-specific enzyme-complemented activatorsorbent assay. Hg(II) was immobilized and presented on a matrix taking advantage of the high affinity of Hg(II) to SH residues. The immobilized Hg(II) could be specifically detected down to the parts-per-billion level by quantifying the β-galactosidase activity of the bound fusion protein complex.     

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