Development of Dot-ELISA for the detection of human rotavirus antigen and comparison with RNA-PAGE

AIMS: Development of a simple, specific, rapid and inexpensive Dot-ELISA test for diagnosis of rotaviral antigen in stool samples. METHODS AND RESULTS: Hyperimmune rabbit antisera raised against SA-11 (Simian Agent-11) strain was used as primary antibody. The secondary antibody conjugate used was the goat anti-rabbit IgG alkaline phosphatase, and BCIP/NBT solution was used as substrate. Faecal extracts were diluted 10-fold and used for the detection of rotavirus antigen. RNA-PAGE was performed to compare the specificity and sensitivity of the diagnostic tests. Dot-ELISA positive samples were further confirmed by Western blot analysis. CONCLUSIONS: This Dot-ELISA test could be used as an alternative method for diagnosing rotaviral samples in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: The Dot-ELISA test is simple, specific, rapid and cost effective. It is suitable for identifying a large number of samples obtained from epidemiological studies and hence, reducing the death rate of rotavirus-infected patients.     

Filter-paper blood samples for ELISA detection of Brucella antibodies in caribou

We evaluated blood collected on Nobuto filter-paper (FP) strips for use in detecting Brucella spp. antibodies in caribou. Whole blood (for serum) and blood-saturated FP strips were obtained from 185 killed arctic caribou (Rangifer tarandus groenlandicus). Sample pairs (serum and FP eluates) were simultaneously tested in duplicate using competitive enzyme-linked immunosorbent assay (c-ELISA) and indirect ELISA (i-ELISA) for Brucella spp. Prior work based on isolation of Brucella spp. revealed sensitivity (SE) and specificity (SP) of 100% and 99%, respectively, for both these serum assays in caribou. Infection status of the animals in the current study was unknown but recent sampling had revealed clinical brucellosis and >40% Brucella antibody prevalence in the herd. To assess the performance of FP relative to serum in these assays, serum was used as the putative gold standard. On both assays, the findings for duplicate runs (A and B) were similar. For c-ELISA run A, the FP Brucella prevalence (47%) was lower than serum prevalence (52%), with SE 89% (95% confidence interval [CI]: 82-95%) and SP 99% (97-100%). For i-ELISA run A, serum and FP Brucella prevalence rates were identical (43%), and the SE and SP of FP testing were 100% and 99% (97-100%), respectively. The findings suggest better FP test performance with i-ELISA than with c-ELISA; however, i-ELISA does not distinguish cross-reacting antibodies induced by Brucella vaccination or exposure to certain other Gram-negative pathogens. Results for duplicate FP eluates (prepared using separate FP strips from each animal) were strongly correlated for both protocols (r=0.996 and 0.999 for c-ELISA and i-ELISA, respectively), indicating minimal variability among FPs from any individual caribou. Dried caribou FP blood samples stored for 2 mo at room temperature are comparable with serum for use in Brucella spp. c-ELISA and i-ELISA. Hunter-based FP sampling can facilitate detection of disease exposure in remote regions and under adverse conditions, and can expand wildlife disease surveillance across temporospatial scales.     

鼠疫耶尔森菌rV抗原单抗系统及其ELISA检测方法的建立

目的建立ELISA双抗体夹心法,测定重组毒力因子rV抗原含量。方法采用杂交瘤技术,制备鼠疫菌rV抗原的鼠单克隆抗体,对抗原表位和单抗特异性进行分析及鉴定,建立ELISA双抗体夹心法,并验证方法的专属性、准确性、精密度和线性范围。结果成功组建了鼠疫菌rV抗原诊断试剂,灵敏度最低检测值为10 ng/mL。结论该方法可用于免疫学检测鼠疫组分疫苗原液rV抗原含量及制备过程中抗原活性,是鼠疫组分疫苗制备中一种重要的质量控制手段,也为进一步开发鼠疫诊断试剂盒及其他相关研究奠定了基础。     

百日咳抗体酶联检测试剂盒的研制及其应用

为了研制百日咳抗体酶联检测试剂盒以调查吉林省2~8岁儿童百日咳、白喉及破伤风抗体水平。采用百日咳菌液经硫酸铵盐析、PBS溶液浸提及蔗糖密度梯度离心提取的PT和FHA作为包被抗原,辣根过氧化物酶(HRP)标记羊抗人IgG,作为抗体制备ELISA试剂盒。并经敏感性、特异性、重复性试验考查该试剂盒质量。结果显示,试剂盒敏感性可达0.0036 IU/m l;重复性较好,CV<4%。试剂盒置于37℃3d敏感性无明显变化。吉林省2~8岁儿童白喉、破伤风的抗体水平较高,百日咳的抗体水平相对较低。此方法制备的百日咳抗体酶联检测试剂盒的质量符合要求,可应用于临床检验与流行病学监测。     

Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

A dot enzyme-linked immunosorbent assay (dot-ELISA) was standardized using excretory-secretory antigens of Toxocara canis for the rapid immunodiagnosis of human toxocariasis. Thirty patients with clinical signs of toxocariasis, 20 cases with other parasitic diseases, and 40 healthy subjects were tested. A total of 0.2 ng of antigen per dot, serum dilution of 1:160 and dilution conjugate of 1:1000 were found optimal. The sensitivity and specificity of the assay were 100 and 95%, respectively. Comparable sensitivity of dot-ELISA and the standard ELISA was obtained, but only 3 cross-reactions occurred in the dot-ELISA, compared with 6 in the standard ELISA. Dot-ELISA is simple to perform, rapid, and low cost. Large-scale screening studies should be done to evaluate its usefulness under field conditions.     

Evaluation of ELISA for detection of Giardia lamblia-specific copro-antigen employing monospecific antibodies

An enzyme linked immunosorbent assay (ELISA) system, using monospecific antibodies for the detection of Giardia lamblia specific 66 kDa copro-antigen has been developed and evaluated. The assay detected the antigen in stool eluates of all the 24 microscopically confirmed cases of giardiasis and in 17 (68%) of the 25 microscopy-negative clinically suspected cases of giardiasis. None of stool eluates from 20 subjects infected with other protozoal/helminthic intestinal parasites or from 20 apparently healthy subjects had G. lamblia-specific copro-antigen. The ELISA employing monospecific antibodies is a sensitive and specific tool for the diagnosis of giardiasis and is especially useful for confirming microscopy-negative suspected cases of giardiasis.     

Chemiluminescence ELISA for the detection of antibodies to bovine leukaemia virus antigens

A chemiluminescence enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine leukaemia virus antigens (BLV) has been developed. The possibility of using an enhanced chemiluminescence reaction for the determination of adsorbed immunoperoxidase conjugates was studied in this work. The intensity of chemiluminescence depends on both the concentration of reagents and experimental conditions used. The efficiency of the assay is determined by the formation of an immobilized antigen monolayer. A relationship between the quantity of the protein added and adsorbed has been shown. The optimal time and temperature for the antigen–antibody incubation steps have been estimated for each system (3h at 37°C was chosen as a standard incubation time). A linear dependence of the chemiluminescence intensity and optical density on the concentration of antibodies to the BLV antigens was observed. The detection limit of antibodies in the chemiluminescence ELISA is 2–3 times lower than that in the spectrophotometric one. The results obtained indicate the possibility of using both methods.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection ofCampylobacter jejuni antibodies,and comparison with a complement fixation test (CFT)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of total anti-Campylobacter immunoglobulins in human sera. In this assay disintegratedCampylobacter bacteria were used as the antigen. Absorption tests including other possibly enteropathogenic bacterial species showed that the ELISA system displayed a high immunological specificity forCampylobacter. Using this ELISA it was found that in about 80% ofCampylobacter patients theseCampylobacter antibodies are produced to almost maximal levels within 8 days after onset of disease, and that they may persist for at least 4 months. Indeed,Campylobacter antibodies were demonstrated at low levels in a large number of control sera. However, accepting an antibody titre of 1: 640 as indicative ofCampylobacter infection, the statistical sensitivity of the ELISA system was 77% and the specificity 95%. In an epidemiological survey a high association was demonstrated between the severity ofCampylobacter-related symptoms and antibody titre values. Assessment ofCampylobacter antibody titres by means of this ELISA and by a complement fixation test in 92 sera from index patients and contacts with and without symptoms showed a high association of results.     

A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin

Fourteen strains of Bacillus cereus isolated from different sources were examined for their ability to produce diarrhoeal enterotoxin by two commercial immunoassay kits (Oxoid BCET-RPLA and Tecra ELISA) and the microslide immunodiffusion assay. One strain that was positive in monkey feedings, as well as a number of other strains isolated from diarrhoeal outbreaks, gave positive results in the ELISA and negative results in the RPLA test systems. When tested in the microslide assay, these strains produced only one antigen which formed a line of identity with the reference toxin. The results of the control toxins provided with the kits substantiated that the two commercial assays did not detect the same antigen. Cultures positive with both assay kits were shown to produce diarrhoeal enterotoxin (by a line of identity) and other antigens in the microslide immunodiffusion assay.     

A comparison of three ELISA systems for the detection of Mycoplasma pulmonis antibody in rats

Barrier maintained and conventionally reared rats were tested for the presence of antibody to M. pulmonis using three ELISA test systems. Agreement between the two commercial test kits was 100% for both the barrier maintained and conventionally reared rats. Agreement between the kits and the non-commercial ELISA was 96% for barrier maintained rats, but only 56% for conventionally reared rats. Procedural differences or the presence of reactive substances in the sera of conventionally reared rats could have accounted for the discrepancy between the non-commercial and commercial tests.     

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

双抗体夹心ELISA法检测肾综合征出血热汉滩病毒糖膜蛋白

用双抗体夹心ELISA法检测肾综合征出血热（HFRS）汉滩病毒LRI株糖膜蛋白（GP）,并用SDS－PAGE电泳对ELISA检测结果进行验证,实验结果显示,双抗体夹心ELISA法特异性强,重复性好,简便易行,是HTN病毒糖膜蛋白较为理想的检测方法,对HFRS疫苗的生产和质量控制有重要意义。     

ELISA test for anti-neutrophil cytoplasm antibodies detection evaluated by a computer screen photo-assisted technique

The computer screen photo-assisted technique (CSPT), a method for substance classification based on spectral fingerprinting, which involves just a computer screen and a web camera as measuring platform is used here for the evaluation of a prospective enzyme-linked immunosorbent assay (ELISA). A anti-neutrophil cytoplasm antibodies (ANCA-ELISA) test, typically used for diagnosing patients suffering from chronic inflammatory disorders in the skin, joints, blood vessels and other tissues is comparatively tested with a standard microplate reader and CSPT, yielding equivalent results at a fraction of the instrumental costs. The CSPT approach is discussed as a distributed measuring platform allowing decentralized measurements in routine applications, whereas keeping centralized information management due to its natural network embedded operation.     

大鼠仙台病毒ELISA、间接免疫荧光和免疫印迹三种检测方法比较

目的比较ELISA（enzyme-linked immunosorbent assay）、IFA（immuno-fluorescence assay）和WB（Western blot）三种方法在大鼠仙台病毒血清学检测中的差异。方法仙台病毒蛋白抗原经凝胶电泳分离转移后用于血清学检测的WB方法;使用IFA、ELISA方法对20份无菌大鼠、227份SPF大鼠以及63份清洁级大鼠送检血清样品进行检测,阳性及可疑样品用WB方法进行了验证。结果 20份无菌大鼠血清样品被3种方法检测为仙台病毒抗体阴性;SPF级大鼠样品被IFA方法判定为阴性,1.32%（3/227）被ELISA方法判定为阳性,其中有2/3被WB确认为阳性;ELISA、IFA和WB在清洁级大鼠样品中检出仙台病毒的阳性率分别为为18.12%、11.34%和15.87%。结论三种检测方法灵敏度从高到低依次为ELISA、WB和IFA。WB方法可作为IFA和ELISA难以确定结果的替代方法。     

A sensitive and specific blocking ELISA for the detection of rabbit calicivirus RCV-A1 antibodies

ABSTRACT: BACKGROUND: Antibodies to non-pathogenic rabbit caliciviruses (RCVs) cross-react in serological tests for rabbit hemorrhagic disease virus (RHDV) and vice versa, making epidemiological studies very difficult where both viruses occur. It is important to understand the distribution and interaction of the two viruses because the highly pathogenic RHDV has been used as a biocontrol agent for wild rabbits in Australia and New Zealand for the past 17 years. The presence of the benign RCV Australia 1 (RCV-A1) is considered a key factor for the failure of RHDV mediated rabbit control in cooler, wetter areas of Australia. RESULTS: A highly sensitive and specific blocking ELISA was developed for the detection of RCV-A1 antibodies. When sera from rabbits with a known infection history for either RCV-A1 or RHDV were tested, this assay showed 100% sensitivity and no cross-reactivity with RHDV sera (100% specificity). CONCLUSIONS: This new ELISA not only allows the detection of RCV-A1 at a population level, but also permits the serological status of individual rabbits to be determined more reliably than previously described methods. This robust and simple to perform assay is therefore the tool of choice for studying RCV-A1 epidemiology in Australian wild rabbit populations.     

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection.