Rapid determination of members of the family Enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen.

An immunological method for the detection of members of the family Enterobacteriaceae in drinking water was developed. The method was based on a sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody immunoglobulin G2a 898 against enterobacterial common antigen. The enterobacterial common antigen sandwich ELISA combined with selective preenrichment culture could be performed in only 24 h. Six hundred sixty-eight water samples from a variety of German public water supplies were screened to verify the effectiveness of the new method. Ninety-eight percent of the results obtained by the immunological method could be confirmed by conventional microbiological methods. The immunological method proved to be considerably faster and more specific and sensitive than the standard method specified by the German drinking water regulations.     

Enterobacterial common antigen: isolation from Shigella sonnei, purification and immunochemical characterization.

In the studies presented the effective procedure of isolation and purification of enterobacterial common antigen from Shigella sonnei has been elaborated. The method is based on sonification of bacterial suspension in the presence of lysozyme and EDTA and subsequent extraction of the pellet with boiling water. The crude extract of common antigen was purified by fractionation with ethanol and chromatography on silica gel and Sephadex LH-20. The comparison of several extraction procedures of enterobacterial common antigen from Shigella sonnei proved that the method described above is most effective. The purified enterobacterial common antigen preparation obtained preserved full biological activity: antigenicity (precipitation and activity in enzyme-linked immunosorbent assay), immunogenicity in rabbits, ability to coat erythrocytes (passive hemagglutination) and inhibitory activity in passive hemagglutination. The pure enterobacterial common antigen was identified to 90% as a polymer of N-acetyl-D-mannosaminuronic acid and N-acetyl-D-glucosamine (2:1, molar ratio), O-acetylated and containing 3.2% fatty acids (C16:0 and C18:1, not oleic). It contains 5.3% nitrogen, less than 4% protein, less than 0.5% phosphorus and less than 1.6% neutral sugar; glycerol and RNA were not found in the preparation.     

Development of a piezoelectric immunosensor for the detection of enterobacteria.

A piezoelectric crystal immunosensor has been developed for the detection of enterobacteria in drinking water using antibodies against the enterobacterial common antigen. Applying an anti-enterobacterial antibody layer via protein A immobilization onto a 10-MHz crystal, a response is observed for 10(6) to 10(9) cells ml-1 of Escherichia coli K12, and for various other antigens of the Enterobacteriaceae family.     

Development of oligonucleotide primers for the specific PCR-based detection of the most frequent Enterobacteriaceae species DNA using wec gene templates

Oligonucleotide primers were designed for the PCR-based detection of the wec gene cluster involved in the biosynthetic pathway leading to the production of enterobacterial common antigen (ECA). Escherichia coli DNA was detected using wec A, wec E, and wec F gene primers. The wec A primers were specific for E. coli. The wec E and wec F primers enabled the detection of the most frequent species of the Enterobacteriaceae found in blood and urine specimens as well as in water. The sensitivity of the assay was approximately 1.2 x 102 bacteria/mL of water. Thus, these primers represent an important step in the molecular diagnosis of major Enterobacteriaceae infections. Their role in the routine testing of contamination in drinking water and food may prove to be very useful. The DNA of Enterobacteriaceae species is detected in a first step PCR, followed by specific identification of important pathogens like E. coli O157, Shigella spp., Salmonella spp., and Yersinia spp.     

Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

Excess of factor X antigen versus clotting counterpart in coumarin treated patients as determined by an Elisa method

Factor X antigen was assayed by means of an Elisa (sandwich) method in 36 patients on long-term anticoagulant therapy. The average value observed was 31.4% +/- 12.2. In almost every instance the antigen level was higher than the clotting counterpart (14.4% +/- 4.5). In a few instances no major difference was noted between factor X antigen and factor X activity. The method correlated fairly well with other immunological methods (electroimmunoassay and Laser Nephelometer). Therefore, factor X Elisa method appears to be a suitable method for factor X antigen evaluation.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.     

The effect of antibodies to the enterobacterial common antigen (ECA) on experimental mouse salmonellosis

Abstract The protective capacity of antibodies to the enterobacterial common antigen (ECA) was tested in experimental mouse salmonellosis after intraperitoneal challenge by moderately virulent smooth Salmonella typhimurium . No evidence could be found for a role of anti-ECA in protection or opsonization in assays in which homologous anti- Salmonella antiserum was strongly positive.     

肠杆菌共同抗原的研究进展

肠杆菌共同抗原(Enterobacterial common antigen,ECA)是由多糖重复单元组成的多聚糖,几乎表达于所有肠杆菌细菌外膜,具有生物学功能。ECA由多基因协同作用而合成,这些基因在肠杆菌细菌基因组上成簇存在,形成ECA抗原基因簇。ECA是重要的毒力因子,在肠杆菌细菌入侵宿主、体内存活等过程中有一定作用。同时,ECA在维持细菌外膜渗透屏障、鞭毛表达、群集运动及抗胆酸胆盐等方面也有重要作用。此外,锚定在细菌脂多糖核心区的ECALPS还是细菌重要的表面抗原,能激发宿主产生高水平抗体,可以作为疫苗研究的靶点。结合笔者的研究,文中对ECA纯化、基因结构和合成、免疫特性、生物学功能及应用等方面进行了综述。     

双单克隆抗体ELISA间接夹心法检测流行性出血热病毒抗原

建立了检测流行性出血热(EHF)病毒抗原的双单克隆抗体(McAb)ELISA同接夹心法,用本法和间接荧光抗体技术(IFAT)相比较,IFAT检出感染细胞内病毒抗原的高峰在感染后第8天,而ELISA检测感染上清中病毒抗原的高峰在第14天,两方法检测179份人工感染EHF病毒的乳鼠脑和肺组织标本,阳性检出率分别为72.1％和68.2％,实验结果表明,本法特异,敏感,简便,不仅可用于EHF病原学研究,也适用于流行病学调查检测大量鼠肺标本。     

Evaluation of Monoclonal Antibody-Based Sandwich Direct ELISA (MSD-ELISA) for Antigen Detection of Foot-and-Mouth Disease Virus Using Clinical Samples

A monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) method was previously developed for foot-and-mouth disease (FMD) viral antigen detection. Here we evaluated the sensitivity and specificity of two FMD viral antigen detection MSD-ELISAs and compared them with conventional indirect sandwich (IS)-ELISA. The MSD-ELISAs were able to detect the antigen in saliva samples of experimentally-infected pigs for a longer term compared to the IS-ELISA. We also used 178 RT-PCR-positive field samples from cattle and pigs affected by the 2010 type-O FMD outbreak in Japan, and we found that the sensitivities of both MSD-ELISAs were about 7 times higher than that of the IS-ELISA against each sample (P<0.01). In terms of the FMD-positive farm detection rate, the sensitivities of the MSD-ELISAs were about 6 times higher than that of the IS-ELISA against each farm (P<0.01). Although it is necessary to conduct further validation study using the other virus strains, MSD-ELISAs could be appropriate as a method to replace IS-ELISA for FMD antigen detection.     

麦长管蚜ELISA检测体系的建立

应用麦长管蚜Macrosiphum avenae(Fabricius)单克隆抗体比较直接、间接和夹心ELISA方法。结果表明,间接ELISA的灵敏度高于直接ELISA,灵敏度分别为0.042μg/mL和0.083μg/mL,而双抗夹心ELISA灵敏度很差,无法使用。建立用于检测捕食性瓢虫肠道内麦长管蚜抗原的间接ELISA检测体系,为进一步应用单克隆抗体评价捕食性瓢虫对麦长管蚜的控制作用奠定可靠的基础。     

Fluorescence immunoassay system based on the use of a pH-sensitive phase-separating polymer

Poly(N-isopropylacrylamide-co-methacrylic acid) [P(NIPAAm-co-MAA)], a linear water-soluble pH-sensitive phase-separating polymer, was synthesized and used as a novel separation carrier for the reactants in immunoassay. This polymer precipitates out of water below a critical pH 5.8 at 37 degrees C and redissolves when the pH of solution is above 6.2. The characteristic of this polymer makes it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. The above approach was applied to determination of alpha-fetoprotein with the competitive immunoassay format. Compared with traditional ELISA using the same reactants, the proposed method was much faster (the assay time decreased from 100-120 to 30 min) and showed similar sensitivity, i.e., 0.04 ng/mL. In addition, a sandwich immunoassay method for the determination of hepatitis B surface antigen was also studied, and the results showed that the pH phase-separating immunoassay could be carried out through a sandwich or a competitive method. This general technique may also be used for a wide variety of separation processes in addition to immunoassay, in which a specific component is to be isolated for analysis, recovery, or disposal.     

旋毛虫Ts87抗原的免疫学特性及保护性的初步研究

应用Western btot和ELISA方法对纯化的重组蛋白PET—28a( )／Ts87进行免疫学特性鉴定及保护性研究。Western blot和ELISA结果显示,Ts87抗原可被人工感染旋毛虫的兔血清、病猪血清、病人血清及抗Ts87的兔血清所识别。Ts87抗原免疫BABL/c小鼠,较对照组减虫率为29％,说明Ts87抗原可作为旋毛虫免疫诊断和疫苗的候选抗原。     

抗Trx单克隆抗体用于血吸虫病诊断的研究

目的：建立双抗体夹心ELISA 法检测日本血吸虫硫氧还蛋白（Thioredoxin,Trx）。方法：用重组日本血吸虫Trx（rTrx）蛋白免疫BALB/c 小鼠,筛选高滴度、高特异性的单克隆抗体建立双抗体夹心ELISA法。通过检测日本血吸虫排泄- 分泌物（excretorysecretions,ES）与rTrx的浓度评价该方法的敏感性;通过对健康人血清的检测确定其特异性;通过对布氏姜片吸虫病、华支睾吸虫病、卫氏并殖吸虫病、囊虫病患者血清进行交叉反应试验,评价该方法的特异性。结果：获得2 株稳定分泌抗rTrx 蛋白单克隆抗体的杂交瘤细胞株,命名为McTrx1 和McTrx2。以McTrx1 为包被抗体,HRP-McTrx2 为酶标抗体,建立的双抗体夹心ELISA 可检测出ES的最低浓度为4.8 μg/ml,检测出rTrx 的最低浓度为1.2 滋g/ml。该方法的特异性为96 %。结论：以抗rTrx 蛋白单克隆抗体McTrx1 与McTrx2为基础建立的双抗体夹心ELISA 法具有较高的特异性。     

Biosynthesis of enterobacterial common antigen requires dTDPglucose pyrophosphorylase determined by a Salmonella typhimurium rfb gene and a Salmonella montevideo rfe gene.

In group C1 salmonellae, rfe and rff genes linked to the ilv locus specify the synthesis of a glycolipid called the enterobacterial common antigen. In contrast, in group B salmonellae the synthesis requires in addition some of the genes in the rfb cluster, the main genetic determinant of the O side chains of lipopolysaccharide. In an effort to define the biochemical functions of these rfb genes, we looked in Salmonella typhimurium LT2 (group B) for rfb mutants in which the synthesis of both enterobacterial common antigen and the O side chains would be blocked in a manner suppressible by the wild-type rfe cluster of S. montevideo, of group C1. We found one mutant with these characteristics. This rfb mutation affected the activity of dTDPglucose pyrophosphorylase (glucose-1-phosphate thymidylyltransferase, EC 2.7.7.24). Whereas the rfe cluster of S. montevideo contained a gene producing this enzyme activity, there was no evidence for the presence of such a gene in the rfe cluster of group B strains. These results also showed that the synthesis of dTDP-glucose is necessary for the biosynthesis of enterobacterial common antigen; this conclusion fits with the recent demonstration of 4-acetamido-4,6-dideoxy-D-galactose as a component of enterobacterial common antigen (Lugowski et al., Carbohydr. Res. 118:173-181, 1983), because the biosynthesis of the donor of this sugar, dTDP-4-acetamido-4,6-dideoxy-D-galactose, requires dTDPglucose pyrophosphorylase.     

Detection of common flagella antigen in Bacillus cereus by monoclonal antibody

Bacillus cereus has been classified into 23 types by immunochemical analyses of flagella antigen, but a common antigenic determinant of flagella had not been determined. When the immunochemical method of classification had changed from "agglutination method" to "enzyme-linked immunosorbent assay (ELISA)," the cross-reactivity between each flagella type was increased. These results indicated that the application of ELISA method would enable detection of the common antigenic determinant of B. cereus. Therefore, we attempted to make monoclonal antibody against common flagella antigen that could be detected by ELISA. Monoclonal antibody provided was specific for B. cereus (H-1 to H-23) and did not show the cross-reactivity with Escherichia coli NIH and Bacillus subtilis.     

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.     

Use of monoclonal antibodies in immunoenzyme analysis for demonstrating antigenic monovalency

To prove the monovalence of the antigen a method has been developed consisting of ELISA with the use of monoclonal antibodies in combination with the antibody neutralization test. Yersinia pestis capsular antigen was disintegrated by heating at 100 degrees C for a short time and subsequently passed through a column packed with Sephadex G-50. The portions of the eluate, showing high activity in the antibody neutralization test and low activity in ELISA (the double antibody sandwich scheme), contained mainly the monovalent antigen. This antigen was replaced by the polyvalent antigen from the antibody complex, but if such complex had been previously fixed by treatment with glutaraldehyde, no replacement of the monovalent antigen by the polyvalent one occurred.