Validation of an ELISA for the diagnosis of recent Campylobacter infections in Guillain–Barré and reactive arthritis patients

Weeks or months following Campylobacter infection, a small proportion of infected individuals develop Guillain-Barré syndrome (GBS) or reactive arthritis (ReA). Stool culture for Campylobacter is often negative in these patients, and serology is therefore the method of choice for diagnosing a recent infection with Campylobacter. This study developed a capture ELISA system to detect anti-Campylobacter IgA and IgM antibodies indicative of a recent infection. The sensitivity of the assay was 82.0% in uncomplicated Campylobacter enteritis patients, 96.2% in GBS patients who were culture-positive for Campylobacter, and 93.1% in culture-positive ReA patients, with a specificity of 93.0%. The assay allows identification of Campylobacter infection in patients with post-infectious neurological and rheumatological complications.     

Enzyme-Linked Immunosorbent Assay for Determination of Major Excrement Allergens of House Dust Mite Species D. pteronyssinus, D. farinae and D. microceras

Species-specific enzyme-linked immunosorbent assays (ELISA) for major excrement allergens (Dp42, Df6 and Dm6) of D. pteronyssinus, D. farinae and D. microceras in house dust were established, using immunoabsorbed, monospecific rabbit antibodies, coupled to horse radish peroxidase. The limit of detection was 13, 4 and 38 ng/ml, respectively. The coefficient of variation for the entire procedure, including dust sieving (212 micron) and extraction was 5-16% for allergen levels above 1000 ng/g dust. Allergen concentration by ELISA correlated well with the number of mite bodies identified and counted by microscopy in 31 dusts (r = 0.88, 0.86 and 0.82 for combined Dermatophagoides sp., D. pteronyssinus and D. farinae group, resp.) Dermatophagoides allergen was recorded in 21/22 mattress dusts (median: 26,000 ng/g; maximum: 290,000 ng/g). D. pteronyssinus allergen occurred in largest amounts (median 7,500 ng/g) followed by D. microceras (median 650 ng/g) and D. farinae (median 240 ng/g).     

An ELISA method for the detection of autoantibodies to adrenal cortex

An enzyme-linked immunosorbent assay (ELISA) is described for autoantibodies to adrenal cortex. Microsomes were prepared from fresh human adrenal glands, and microtitre ELISA plates were incubated at 4 degrees C overnight with 25 micrograms antigen/ml, the optimal concentration for the system. Optimal dilution of patient's serum was 1/500. Peroxidase-labelled anti-human IgG and IgM sera were used in separate tests and o-phenylenediamine and H2O2 served as substrate. Intra-assay variance of optical density units was 4.5%, and inter-assay variance was negligible when antigen preparations from 2 different adrenal glands were compared. All sera positive or negative at first test gave the same qualitative result in a second. Non-organ-specific binding of sera containing mitochondrial or ribosomal antibodies was eliminated by a blocking ELISA system where the antigen-coated plates were incubated with test sera, and in a second step, peroxidase-labelled IgG from an adrenal antibody-positive control serum was added. In this test, optimal antigen concentration for coating of plates was 6.25 micrograms/ml and optimal serum dilution 1/50. The ELISA proved more sensitive and reproducible than indirect immunofluorescence. Adrenal antibodies detected by ELISA were usually of IgG class alone and only 1 of the 30 positives also contained IgM specificity. 30 out of 38 sera (79%) from patients with 'idiopathic' Addison's disease were positive whereas immunofluorescence revealed only 23 (61%) at first testing and another 4 positives when sera were tested on different adrenal glands. The ELISA described is useful for both scientific work and clinical diagnosis of autoimmune adrenalitis.     

重组猪戊型肝炎病毒ORF2区主要抗原域的原核表达及鉴定

目的：在大肠杆菌中表达猪戊型肝炎病毒ORF2区主要结构蛋白,并对其进行血清学鉴定。方法：通过RT-PCR技术从一份猪粪中扩增并克隆戊型肝炎病毒主要的结构基因片段,将该片段插入pET-32a表达载体,在原核系统中融合表达蛋白,Western blot和间接ELISA方法分析该蛋白的抗原性。结果：SDS-PAGE分析结果表明,获得45kD的目的蛋白,该重组蛋白可以与HEV阳性血清反应,而不与阴性血清反应,表明该蛋白具有良好的抗原性。结论：本研究获得了猪戊型肝炎病毒重组抗原,可与HEV阳性血清产生特异性的结合反应,可以作为诊断用的重组蛋白,为研制敏感、特异的HEV诊断试剂盒,行之有效的HEV基因工程疫苗及为HEV感染的预防和临床治疗提供资料。     

Production and characterization of antibody against fusarochromanone

Antibody against fusarochromanone (TDP‐1) was obtained from rabbits after immunization with TDP‐1 conjugated to bovine serum albumin (BSA). An indirect enzyme‐linked immunosorbent assay (ELISA) using TDP‐1‐ovalbumin conjugate as the antigen coated on to the microtiter plate was used for monitoring the antibody liter. For toxin detection, a direct competitive ELISA in which TDP‐1 was conjugated to horseradish peroxidase (HRP) was used. Competitive direct ELISA revealed that the antibody had about 5.6 and 4.5 times greater binding efficiency for monoacetyl fusarochromanone (TDP‐2) and diacetylated TDP‐1 than TDP‐1. The concentration causing 50% inhibition of binding of TDP‐1‐HRP to the antibody by TDP‐1, TDP‐2 and diacetyl‐TDP‐1 were 2.8, 0.5 and 0.62 ng/ml, respectively. For the analysis of fusarochromanones in wheat and barley, the toxins were first extracted from the commodities with 100% methanol. A small aliquot of the extract was dried, acetylated, diluted in buffer and then analyzed directly by ELISA. The overall recovery for fusarochromanone in the wheat and barley samples spiked with TDP‐1 in the concentration range of 20 to 500 ppb were found to be 97% and 103.4% with cv of 15% and 11.2% for barley and wheat, respectively.     

Purification of biologically active rubella virus antigens by immunoaffinity chromatography

A general procedure for isolating biologically active rubella virus antigens (VPI, Mr = 61,000; VP2, Mr = 45,000; VP3, Mr = 36,000) by monoclonal antibody affinity chromatography is described. Complexes formed between monoclonal antibodies and rubella virus antigens were found to be stable either at low pH or in Tris buffer containing detergent and high salt, but were efficiently dissociated by 5% diethanolamine, pH 11.5, or 50 mM lithium diiodosalicylate buffer, pH 8.0. Chromatographically purified rubella viral antigens retained their antigenicity as determined by enzyme-linked immunosorbent assays. Biological studies showed that rubella structural proteins VP2 and VP3 had no hemagglutinin function while the mixture of VP1 and VP2 and VP3 directly demonstrated hemagglutination activity. These results indicate that VP1 is at least in part responsible for the hemagglutinin function of rubella virus.     

抗慢性B淋巴细胞白血病患者IgM单抗相对亲和力的分析

本文采用间接ELISA法检测了11株抗B-CLL患者IgM腹水和杂交瘤上清McAb的相对亲和力。结果提示,各株McAb结合抗原的相对亲和力不同。根据50％最大结合浓度分为两组。其中4株杂交瘤上清McAb与纯化腹水McAb测得结果基本吻合。实验结果为合理地选用这些单抗提供了重要依据。     

Enzyme-linked immunosorbent assay (ELISA) for mumps virus antibodies

An ELISA for the detection of mumps virus-specific IgG and IgM antibodies was developed. Three different antigen preparations were compared. Equally good results were obtained with virus concentrated by ultracentrifugation and with virus that was further purified by sucrose gradient centrifugation. Crude infected allantoic fluid was unsuitable for use as antigen. The variability and reproducibility of the tests were satisfactory. When the ELISA was compared to conventional serological methods, a good correlation of IgG absorbance values with complement fixation (CF) antibody titers was found (r = 0.574), the ELISA being more sensitive in detecting antibodies in acute-phase sera. For the determination of immunity, ELISA IgG values were compared with results obtained in hemagglutination-inhibition (HI) and hemolysis-in-gel (HIG) tests. Again there was a good correlation with both tests (r_HI = 0.528, r_HIG = 0.667). The ELISA was more sensitive than the HI and HIG test for the detection of low levels of antibodies.     

Immobilization of antibodies on a new solid phase for use in ELISA

Antibodies to myoglobin were immobilized by covalent linkage to polyester film for use in a solid-phase ELISA. The covalent linkage of antibody to this new solid phase was accomplished by partial acid hydrolysis of the film followed by periodate oxidation. About 60 ng of protein could be immobilized per cm2 of film and the binding was stable. Antimyoglobin IgM immobilized on the films was used in the ELISA technique to detect myoglobin within the range 0.25-10 ng. The assay procedure was found to be very accurate and the coefficient of variation of each concentration ranged from 0.63 to 2.1%. Furthermore the immobilized film could be re-used after dissociating the antigen antibody complexes.     

Application of an aldrin and dieldrin ELISA to the detection of pesticides in eggs

This paper details the results obtained when a number of egg samples, collected in Egypt from different races of chicken were analyzed for the presence of the organochlorine insecticides aldrin and dieldrin. A simple ELISA was used for the detection and quantification of aldrin and dieldrin. The test was modified for application in this high protein system. Pesticide was detected in 83–352% (14/17) of the samples at levels ranging from 0.006 to 0.7 ppm (0.006 to 0.7 μg ml^‐1). People eating eggs containing these amounts of pesticides which are above the World Health Organisation average daily intake levels would be at increased risk.