Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.
This study describes the use of an IgM capture ELISA using cell culture derived antigens and a polyclonal rabbit antiflavivirus antisera for the detection of dengue positive cases. The IgM capture ELISA is compared with the dot enzyme immunoassay and the results are discussed in the context of dengue endemicity.
Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.
Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6M urea for the first wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n=16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p<0.05) over the first 24 weeks. In Week 4, all animals had avidity values <35% and by Week 8, 8/16 animals had avidity values of >35%. These results suggest that an avidity value of <35% indicates a recent primary infection while a value of >35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody influence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep.
An IgG4 ELISA based on a novel recombinant antigen was evaluated for detection of Brugia malayi infection, using 2487 sera from various institutions: 2031 samples from Universiti Sains Malaysia, 276 blinded sera from 2 other institutions in Malaysia, 140 blinded sera from India and 40 blinded sera from Thailand. These sera were from various groups of individuals, i.e., microfilaraemics, chronic patients, endemic normals, non-endemic normals and individuals with other parasitic and bacterial infections. Based on a cut-off optical density reading of 0.300, the IgG4 ELISA demonstrated specificity rates of 95.6-100%, sensitivity rates of 96-100%, positive predictive values of 75-100% and negative predictive values of 98.9-100%. These evaluation studies demonstrated the high specificity and sensitivity of this test for the detection of active B. malayi infection. Thus, the IgG4 ELISA would be very useful as a tool in diagnosis and in elimination programmes for brugian filariasis.
An in-house enzyme-linked immunoabsorbant assay (ELISA) for SP-A was successfully developed using in-house polyclonal anti SP-A and a commercial polyclonal anti-rabbit immunoglobulin horseradish peroxidase conjugate system. The standard curve, generated by using 50 ng of SP-A to coat the plate and 1:500 dilution of polyclonal anti SP-A as a primary antibody, was linear for concentrations of SP-A ranging from 4 micrograms/l to 4000 micrograms/l and reproducible. Results of recovery study of SP-A from a known sample of tracheal aspirate ranged from 94%-114%. Intra- and inter-assay coefficients of variations were 2.7% and 5.6% respectively for a known sample of tracheal aspirate. Interference study showed that tracheal aspirate did not interfere with the assay. The assay developed was intended to be used for SP-A measurement in tracheal aspirates obtained from neonates with and without respiratory distress syndrome.
This study describes the status of viral encephalitis in Perak, Malaysia during the year 1990. In addition, 14 cases selected from Penang and Perak during the years 1989 and 1990 are presented, with data showing titers of neutralizing antibodies against Japanese encephalitis virus (JEV) and dengue 2 virus, titers of antibodies against JEV and dengue virus antigens as determined by DEIA, and a comparison of these with the presence of IgM to JEV and dengue virus. These data show that there probably is far more viral encephalitis due to JEV in Malaysia than the national figures reflect.
A serological investigation for human T cell leukemia virus I (HTLV-I) infection was carried out at the University Hospital, Kuala Lumpur. A total of 626 sera from a non-patient population and 1,038 sera from unselected in-patients were screened for HTLV-I antibodies using an enzyme-linked immunosorbent assay (ELISA). 27/1664 (1.6%) were found to be reactive. However, on Western blotting, only 2 sera were confirmed positive, both showing reactions for the major core (p19 and p24) and the envelope (gp46) proteins. Both of the serum samples were from unselected hospital patients. Most of the remaining sera which were reactive on screening showed indeterminate results on Western blotting. These were further tested by radioimmunoprecipitation assay (RIPA) and none of these sera gave a positive reaction. Therefore, only 2/1038 (0.19%) unselected patients could be confirmed to have antibodies to HTLV-I. None of the normal individuals screened showed a positive Western blot result. Our data indicate that HTLV-I infection is present in our population, but at a low prevalence rate.
A monoclonal antibody (MAb) based solid-phase blocking ELISA was developed for detection of antibodies to Nipah virus. The ELISA was designed to detect remaining antigens on the plate with anti-Nipah MAb conjugate after the reaction with sample serum, and enabled simple procedure, detection of neutralizing antibody to Nipah virus, and application of samples from different animal species. Forty of 200 swine reference sera examined were positive by the ELISA, of which thirty seven were found positive by serum neutralization test. Sera from a total of 131 fruit bats captured in Malaysia were also tested and all found negative by the both tests. It is considered that the solid-phase blocking ELISA can be used as a screening test for Nipah virus infection followed by the serum neutralization test as confirmatory test.
Because blocking agent occupies most binding surface of a solid phase, its ability to prevent nonspecific binding determines the signal-to-noise ratio (SNR) and reliability of an enzyme-linked immunosorbent assay (ELISA).
The aim of the study was to look into the epidemiology of serodiagnosed cases of leptospirosis at the University Hospital and compare two commercial ELISA Assays to the Microscopic Agglutination Test (MAT). Demographic data for all serodiagnosed cases for the years 1991-1997 were collected. From this data, 104 sera (n = 104) were selected as samples for comparative evaluation of the commercial ELISAs (INDX Dip-S-Ticks and PanBio ELISA) to the MAT test. Thirty two (n = 32) negative control sera were selected from serodiagnosed cases of other differential diagnosis of leptospira infection. The MAT test is a standard test that detects agglutination antibodies to leptospira biflexa, while the INDX Dip-S-Ticks is an ELISA dot test assaying for total anti-leptospira antibodies. The PanBio ELISA is a colorometric assay in test well strips to detect anti-leptospira IgM. The sensitivity, specificity, and efficiency of tests were calculated at a MAT cut-off value of 1:320. Demographic data showed that leptospirosis peaks during March-May and Aug-Nov coinciding with the inter-monsoon period with more men being infected than women and more adults than children. The sensitivity, specificity, and efficiency of test for the INDX Dip-S-Ticks were 83.3%, 93.8% and 87.5% while the values for the PanBio ELISA were 54.2%, 96.9% and 71.3%. The suboptimal PanBio result could be related to the blocking effect of high IgG titres or could be related to the diagnostic MAT cut-off values used in this study. The data hence reflects a pattern of transmission that is related to "wet" occupational risk factors. The commercial assays evaluated, are easier to perform but interpretation of results should be based on level of endemicity. The INDX Dip-S-Ticks allows this flexibility and is a practical alternative to the MAT test.