Early and rapid detection of enterovirus 71 infection by an IgM-capture ELISA

Enterovirus 71 infection is more likely to induce severe complications and mortality than other enteroviruses. Laboratory diagnosis of enterovirus 71 in Taiwan still relies mainly on conventional virus isolation techniques that often require 5-10 days to obtain a result, thus hindering seriously the subsequent treatment and disease control measures. This study was to find a better alternative by developing a rapid enterovirus 71 detecting procedure, which can afford an earlier diagnosis and a more rapid outcome. In this study, an enterovirus 71-IgM-capture enzyme-linked immunosorbent assay (ELISA) was designed and tested with a total of 336 serum specimens collected from 236 cases of reported enterovirus infection with complications. Using virus isolation and neutralization test as standards, the sensitivity and specificity of the new protocol were 97.7 and 93.3%, respectively. Most of the IgM positive serum specimens were collected within 7 days after the onset of symptoms, while it appeared detectable up to 94 days after the onset of symptoms. Apart from being highly sensitive, rapid and low in cost, the new IgM-capture ELISA is sufficiently accurate to provide also reliable results for early detection of the virus. With this protocol, enterovirus 71 infections can be detected within 4h.     

Validation of IgG-sandwich and IgM-capture ELISA for the detection of antibody to Rift Valley fever virus in humans

Rift Valley fever (RVF) virus is an important zoonotic and a potential biothreat agent. This paper describes validation of sandwich and capture enzyme-linked immunoassays (ELISA) based on gamma-irradiated antigens for the detection of RVFV-specific IgG and IgM antibody in humans. Validation data sets derived from testing field-collected sera from Africa (n=2400) were dichotomised according to the results of a virus neutralisation test. In addition, sera from laboratory workers immunized with inactivated RVF vaccine (n=93) and serial sera (n=3) from a single RVF case were used. ELISA data were expressed as percentage of high-positive control serum (PP). Cut-off values at 95% accuracy level were optimised using the misclassification cost term option of the two-graph receiver operating characteristics analysis. During the routine use of assays there was no evidence for excessive intra- and inter-plate variations within and between runs of assays. At a cut-off of 13.2 PP the sensitivity of the IgG-sandwich ELISA was 100% and specificity 99.95%, while for the IgM-capture ELISA the values were 96.47 and 99.44%, respectively, at a cut-off of 7.1 PP. Compared to the virus neutralisation test, the IgG-sandwich ELISA was more sensitive in detection of immunological responses in vaccines. Following natural infection class-specific antibodies were detected in serum taken 6 days after onset of symptoms. The results demonstrate that both assays will be useful for early diagnosis of infection, epidemiological surveillance and for monitoring of immune response after vaccination. As highly accurate, robust and safe tests, they have the potential to replace traditional diagnostic methods which are unable to distinguish between different classes of immunoglobulins, and pose health risks necessitating their use being restricted to high containment facilities outside RVF endemic areas.     

Improved ELISA conditions for detection of plant viruses

Clover yellow vein virus (CYVV) and homologous antisera were used to test effects of time and temperature on enzyme-linked immunosorbent assay (ELISA) in polystyrene substrate plates. Replicated lattice square and Youden square experimental designs were used to measure and account for variation in absorption values associated with sample position within polystyrene plates. Adsorption of coating antibody to polystyrene was relatively rapid, reaching optimum assay efficiency in 1 h at 5°C when applied at 2.5 μg/ml. Binding of antigen and enzyme-linked antibody (conjugate) in their respective steps during ELISA was also rapid. Incubation of antigen and conjugate for 2 h each was adequate to enable detection of 20 ng CYVV in a 100 μl sample, but longer incubation of either reactant improved results. At this virus concentration, reduction in antigen incubation time by one-half could be compensated by doubling the conjugate incubation time and vice versa. Incubation of conjugates at 5°C rather than 30°C increased final ELISA readings (A_400nm) more than two-fold. Substrate hydrolysis followed classic first order kinetics at room temperature. Greater efficiency of late antisera in ELISA was demonstrated by comparison of antisera produced relatively early and late during a rabbit's immune response. Alfalfa mosaic virus and peanut stunt virus with their homologous antisera were used to test the effects of antigen and conjugate incubation times for optimum assay efficiency. The results of these time course experiments with both viruses were similar to those obtained with CYVV. These time and temperature effects on ELISA should be applicable to most rabbit serum-virus combinations.     

Development and evaluation of an IgM capture enzyme immunoassay for diagnosis of recent Norwalk virus infection

A capture enzyme immunoassay specific for Norwalk IgM class antibody was developed. The assay was moderately sensitive, identifying 33/53 (62%) of patients with naturally acquired Norwalk virus infection and 17/18 (94%) of experimentally infected volunteers. The assay was also specific for IgM class antibody and acute Norwalk virus infection and results were generally reproducible. A specific IgM response correlated with seroconversion by total antibody blocking assay and occurred independently of clinical symptoms. Among 81 symptomatic cases composing seven Norwalk outbreaks, specific IgM was absent from acute phase sera collected less than or equal to three days post onset, and was uncommon in sera collected within one week and after five weeks, with an optimal collection time at about two to three weeks. The Norwalk IgM capture immunoassay may be used to augment paired sera assays in the identification of Norwalk-associated outbreaks of acute gastroenteritis.     

Laboratory and field studies of an antigen capture ELISA for bluetongue virus

An improved bluetongue antigen capture ELISA (BTACE) technique was evaluated for its ability to detect the full range of 24 bluetongue (BLU) serotypes. The BTACE detected all 24 serotypes in cell culture fluids, including eight serotypes where the representative strains originated from both Australia and also from the South African reference collection. The amount of infectious virus required to obtain a positive BTACE result varied between 100-1000 TCID50. This was approximately 10-fold more sensitive than the antigen capture test described previously (Hosseini, M., Hawkes, R.A., Kirkland, P.D., Dixon, R., 1998. J. Virol. Methods 75, 39-46.). The BTACE method was compared with conventional passage in cell culture to detect the presence of virus in the tissues of embryonated chicken eggs (ECEs) which had been inoculated intravenously with the blood of sheep and cattle infected experimentally with the eight Australian serotypes of BLU (1, 3, 9, 15, 16, 20, 21, and 23). The BTACE method was at least as sensitive as the conventional cell culture detecting virus in ECEs, obviating the need for prolonged cell culture passage to detect the virus. A comparison of the amount of antigen detected in different embryo tissues indicated that liver homogenates gave the highest positive to negative ratios in the BTACE and were selected as the specimen of choice. In studies of sheep infected with all 24 South African reference BLU serotypes this new BTACE was able to detect viraemia with all serotypes. Finally, the BTACE was validated in surveillance programs for BLU in both New South Wales, Australia and in Yunnan Province, People's Republic of China. Blood samples from sentinel cattle were inoculated into ECEs. Homogenised ECE livers were tested by BTACE and those positive were passaged subsequently in cell culture for virus isolation and identification. This protocol led to the efficient isolation of field isolates of many serotypes. The high sensitivity and broad reactivity of the method indicates that it should be valuable for BLU diagnosis and surveillance programs.     

Development and evaluation of an enzyme-linked immunosorbent assay for use in the detection of bovine tuberculosis in cattle

As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at ～90 to 100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacterium spp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing.     

Development and evaluation of an antibody capture ELISA for detection of IgG to Epstein-Barr virus in oral fluid samples

The development of an EBV IgG antibody capture ELISA (GACELISA) for detection of EBV viral capsid antigen specific IgG in oral fluids is described. The assay was optimised and evaluated using paired serum and oral fluid samples from healthy laboratory staff (n=82) and oral fluids collected either for routine measles, mumps, and rubella testing (n=629) or for an epidemiological study of atopic dermatitis (n=252). Statistical analysis by mixture modelling was used to determine the GACELISA cut-off and to estimate the sensitivity and specificity of the assay. Sensitivity and specificity was also assessed by comparing the results of immunofluorescence assay for EBV specific IgG in serum with those of GACELISA in 82 matching oral fluids. Compared to serum immunofluorescence assay, oral fluid GACELISA was found to have a sensitivity of 82.2 and 88.9% specificity with these samples. Mixture modelling, predicted the GACELISA to be 88.4% sensitive and of 99.4% specific. The prevalence of antibody to EBV in oral fluids was found to be 73.8% in laboratory staff, 34.4--73.9% in measles, mumps, and rubella patients and 22.2% in atopic dermatitis study participants. A robust, reliable and reproducible EBV GACELISA has been developed which will be a useful tool for epidemiological investigations.     

Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody

An IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) is used widely for serodiagnosis of dengue. A dengue IgM-ELISA with higher sensitivity has been developed. In the new ELISA, anti-dengue IgM antibody, which had been captured on the solid phase, was reacted with tetravalent dengue viral antigens, and detected by a flavivirus group specific monoclonal antibody, D1-4G2-4-15 (4G2). Reaction of 4G2 to viral antigens was similar to that of dengue patients' IgG. Non-specific reaction of 4G2 to the control antigen, which was prepared from uninfected cell culture fluid of mosquito C6/36 cells, was much lower than that of patients' IgG. Thus, specificity of the ELISA with 4G2 was much higher than that with patients' IgG, and lower levels of specific IgM was detected in the serum samples. These results suggest that the modified dengue IgM-ELISA with monoclonal antibody 4G2 has many advantages over the original "in-house" ELISA.     

Development of an ELISA for detection of antibodies to avian reovirus in chickens

An enzyme-linked immunosorbent assay (ELISA) using the expressed sigmaC and sigmaB proteins which induce neutralizing antibodies as the coating antigen (sigmaC-sigmaB-ELISA) for the detection of antibodies to avian reovirus in chickens was developed and compared with serum neutralization and conventional ELISA tests. These assays were used to examine the sera from chickens vaccinated experimentally and farm chickens. The correlation rate between serum neutralization and a sigmaC-sigmaB-ELISA was 100% (156/156), and that between serum neutralization and conventional ELISA was 89.1% (139/156). The results revealed that preparation of an ELISA by using sigmaC and sigmaB of ARV as the coating antigen in detecting the field chicken sera in comparison with the conventional ELISA gave a titer more correlated to the serum neutralization test. The sigmaC-sigmaB-ELISA showed a higher correlation with the serum neutralization-positive and -negative sera than that obtained with conventional ELISA. This combination antigen may thus be the best suited for preparing an ELISA for improving the determination of the immune status of chicken flocks or for detection of chicken infections with avian reovirus.     

Development of an indirect competitive ELISA for the detection of acenaphthene and pyrene

Polycyclic aromatic hydrocarbons (PAHs) have mutagenic and carcinogenic properties. Acenaphthene and pyrene were members of 16 PAHs which were listed as the priority pollutants in water environment by US Environmental Protection Agency. The reported instrumental methods and immunoassays did not meet the need for simple and sensitive detection of acenaphthene and pyrene. In this study, a monoclonal antiobody having high affinities with acenaphthene and pyrene was produced and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for sensitive detection of acenaphthene and pyrene in water sample. The linear range of the assay was between 3.53 and 41.98?ng?mL^?1. The sensitivity was 12.17?ng?mL^?1 which was more sensitive than those of reported ELISAs. The average recovery of acenaphthene and pyrene from three kinds of water samples was 99.08% and 98.45, respectively. The developed ELISA could be used for sensitive detection of acenaphthene and pyrene in water samples.     

快速检测游离前列腺特异性抗原 ELISA 方法的建立

 目的 建立快速检测人血清中游离前列腺特异抗原（f-PSA）的 ELISA 方法。 方法 利用抗 f-PSA 的单克隆抗体（单抗）杂交瘤细胞株，一株单抗 2D1 用于固相包被，另一株单抗2E4进行辣根过氧化物酶标记，采用二步法，建立定量测定人血清 f-PSA 的双抗体夹心ELISA法。对该法的敏感度、精密度、准确性、特异性进行了分析。应用该法与瑞典 CanAg 公司的f-PSA ELISA 试剂盒同时检测了 18 例前列腺癌和 25 例前列腺增生患者血清标本的 f-PSA 含量，进行了两种方法检测结果的相关性分析。 结果 以双抗体夹心 ELISA 法检测 f-PSA，在 f-PSA 0 ~ 20 μg/L 范围内线性良好；敏感度为 0.025 μg/L；批内变异系数为 4.5% ~ 6.2%，批间变异系数为 3.9% ~ 7.2%；回收率为 94.3% ~ 111.1%；与 α1 抗糜蛋白酶结合的结合型PSA 交叉率为 0.7 %；检测 43 份临床标本 f-PSA 含量的结果与瑞典 CanAg 公司 f-PSA 试剂盒检测结果的相关系数为 0.995。 结论 所建立的双抗体夹心 ELISA 方法是一种特异、敏感、简便实用的 f-PSA 快速检测方法，有助于在 PSA 低水平升高的重叠范围内（4 ~ 10 μg/L）鉴别前列腺增生与前列腺癌。     

Development of an ELISA for the detection of autoantibodies to BP230

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the transmembrane hemidesmosomal protein BP180/collagen type XVII and the intracellular plaque protein BP230. The aim of the present study was to develop an ELISA system for the detection of circulating autoantibodies to BP230. We generated five overlapping cDNA constructs covering the entire length of BP230 and expressed them in baculovirus-infected Sf21 insect cells. ELISA reactivity against BP230 was found in 63% of 56 BP patients' sera; the specificity of the ELISA was 93%. Epitope mapping studies showed that the fragment representing the C-terminal portion of BP230 was by far the most frequent target within the molecule. This ELISA provides a useful tool for the detection of autoantibodies to BP230 in BP and other diseases associated with an autoimmune response to this protein.     

A comparison of ELISA and histopathology for the detection of paratuberculosis in dairy cattle

Johnes disease or paratuberculosis is an infectious disease caused by Mycobacterium paratuberculosis and has been described in numerous ruminate species. In cattle, intractable diarrhea, emaciation, and hypoproteinemia in animals older than 19 months characterize the disease. The objectives of this study were to determine the association between severity of histological enteric lesions and the serum absorbed enzyme-linked immunosorbent assay (ELISA) antibody test and comparative efficacy of absorbed ELISA antibody test with histopathological finding of naturally acquired paratuberculosis in the infected dairy cattle of Iran. Fifty mature dairy cows, culled from four M. paratuberculosis-infected herds in Iran were enrolled in this study. Poor physical condition was the major criteria for selection of the cows. Prior to slaughter, blood samples were collected from jugular vein from each animal. All cattle were inspected after slaughter. and samples from 50 cases that showed thickening of different parts of the intestine and enlargement of mesenteric lymph nodes were obtained for histopathological examination. At microscopic examination, specific histopathological characteristics of paratuberculosis were observed in 19 cases. Tuberculoid reactions were observed in five cases, and lepromatous reactions were observed in 14 cases out of the total. Of the total, 19 samples had histopathological finding of paratubercolosis, and only eight animals exhibited a positive antibody response when subjected to an ELISA assessment. The sensitivity, specificity, positive predictive value, and negative predictive value for absorbed ELISA was 42.1%, 100%, 100%, and 73.8%, respectively.     

Development of an ELISA for the detection of chlortetracycline,oxytetracycline and tetracycline residues

A ‘generic’ enzyme immunoassay (ETA) has been developed which is capable of detecting chlortetracycline, oxytetracycline and tetracycline with a limit of determination well below the current maximum residual limit of 600 ng g^‐1 kidney^‐1. The EIA requires no prior sample clean‐up or concentration, being applied directly to tissue homogenate, and will also detect tetracyclines in milk up to 15 ng ml^‐1.     

Use of an immunomagnetic separation‐ELISA technique for the fast detection of growth promoters in cattle urine

The detection of illegal growth promoters in cattle urine by conventional immunoassays, such as radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) has been extensively described. However, improved speed and simplicity, with the development of a fast and simple on‐site test as the ultimate goal, remains an interesting immunochem‐ical challenge. This paper describes two approaches to this problem. To avoid time‐consuming sample preparations, attempts were made to omit the extraction and hydrolysis step. Secondly, instead of the classical microtiter plate as the solid phase, magnetizable beads, which provided a solid phase that was dispersed throughout the sample, were used. In this design, the diffusion limitations of the free immunoreagents to the solid‐phase‐bound immunoreagent (which slow down the kinetics of the antibody‐antigen reaction) were circumvented. This principle was applied to the detection of the β‐agonist clenbuterol and the anabolic steroid nortestosterone in cattle urine. The assay of unextracted urine for the presence of nortestosterone failed due to matrix effects. For clenbuterol, a magnetic particle‐based immunoassay on unextracted urine samples was developed. The test, which can be performed within 90 min using pre‐coated beads, compared favourably to microtitre plate ELISA (with a4h incubation) and to a commercial RIA. The results on test samples were confirmed by gas chromatography/mass spectrometry.     

Development of ELISA for melamine detection in milk powder

A sensitive ELISA method was developed based on a monoclonal antibody to detect melamine in milk powder. Two haptens were prepared using the melamine structural analogue, 2-chloro-4, 6-diamino-1, 3, 5-triazine. The obtained antibody belonged to the IgG1κ family and had high affinity for melamine (affinity constant was 3.09×10⁹). The antibody could also recognise cyromazine (cross-reactivity was 131%) and had low cross-reactivity toward the haptens. The effects of pH, ionic strength and organic solvents on ELISA performance were evaluated. The lowest IC₅₀ value (6.0±0.55 ng/mL) and the limit of detection in milk powder, 50 ng/g, were achieved under optimised conditions. Milk powder spiked at two levels of melamine (100 ng/g and 500 ng/g) was analysed with this ELISA method. Good recovery was obtained (76.2±7.3% at the 100 ng/g spiked level and 91.4±5.0% at 500 ng/g).     

Serological detection of exposure to Cryptosporidium parvum in cattle by ELISA and its evaluation in relation to coprological tests

We evaluated serum examination as an alternative to fecal analysis for the diagnosis of exposure to Cryptosporidium parvum in cattle. The accuracy of the serum ELISA was compared to the combined results of concentration flotation microscopy and fecal enzyme immunoassay. The expected performance of the serum ELISA at different levels of infection with C. parvum was evaluated using the predicative values positive and negative. Optimal conditions for the serum ELISA can be achieved by diluting the serum samples 1:20 and the conjugate 1:8,000. The serum ELISA had a relatively high sensitivity of 97.5% (95% CI=87-100%) and poor specificity, 4% (95% CI=1-20%). There was a poor agreement between the serum ELISA and the fecal tests (kappa=0) on samples collected from adult cows in a high-risk and a low-risk population. Examination of some of these fecal samples using a PCR detection method demonstrated the presence of C. parvum DNA in 10% of the samples.