IgG Capture Enzyme-linked Immunosorbent Assay Research Articles

Comparing the performance of dengue virus IgG and IgG-capture enzyme-linked immunosorbent assays in seroprevalence study

BackgroundDengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. Currently Dengvaxia, the first dengue vaccine licensed in 20 countries, was recommended for DENV seropositive individuals aged 9–45 years. Studying dengue seroprevalence can improve our understanding of the epidemiology and transmission dynamics of DENV, and facilitate future intervention strategies and assessment of vaccine efficacy. Several DENV envelope protein-based serological tests including IgG and IgG-capture enzyme-linked immunosorbent assays (ELISAs) have been employed in seroprevalence studies. Previously DENV IgG-capture ELISA was reported to distinguish primary and secondary DENV infections during early convalescence, however, its performance over time and in seroprevalence study remains understudied.MethodsIn this study, we used well-documented neutralization test- or reverse-transcription-polymerase-chain reaction-confirmed serum/plasma samples including DENV-naïve, primary and secondary DENV, primary West Nile virus, primary Zika virus, and Zika with previous DENV infection panels to compare the performance of three ELISAs.ResultsThe sensitivity of the InBios IgG ELISA was higher than that of InBios IgG-capture and SD IgG-capture ELISAs. The sensitivity of IgG-capture ELISAs was higher for secondary than primary DENV infection panel. Within the secondary DENV infection panel, the sensitivity of InBios IgG-capture ELISA decreased from 77.8% at < 6 months to 41.7% at 1–1.5 years, 28.6% at 2–15 years and 0% at > 20 years (p < 0.001, Cochran-Armitage test for trend), whereas that of IgG ELISA remains 100%. A similar trend was observed for SD IgG-capture ELISA.ConclusionsOur findings demonstrate higher sensitivity of DENV IgG ELISA than IgG-capture ELISA in seroprevalence study and interpretation of DENV IgG-capture ELISA should take sampling time and primary or secondary DENV infection into consideration.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection

IntroductionSevere fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. MethodsTo develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. ResultsThe MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. ConclusionsThe MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Evaluation of Commercially Available Assays for Diagnosis of Acute Dengue in Schoolchildren During an Epidemic Period in Medellin, Colombia.

During an active surveillance study in school children in Medellin, we assessed the performance of two diagnostic strategies for dengue virus. A total of 41 patients with suspected dengue acute infection were evaluated. Diagnostic strategies consisted of one combining Panbio(®) Dengue virus IgM and IgG Capture ELISAs (enzyme-linked immunosorbent assays) with reverse transcriptase polymerase chain reaction (RT-PCR) and another using a commercial rapid SD Bioline Dengue Duo (IgG/IgM + NS1 Ag) test. These two strategies were compared with the enzyme-linked immunospot microneutralization test (ELISPOT-MNT). The sensitivity and specificity were 53.9% and 80.0% for the combination of Panbio(®) ELISAs and RT-PCR tests, and 30.8% and 73.3% for the SD Bioline Duo test, respectively. ELISPOT-MNT detected 16.4% additional cases and revealed the presence of neutralizing antibodies in all the acute samples, evidencing that they were all secondary infections. In contrast, Panbio(®) and SD Dengue Duo rapid tests only classified 23.0% and 26.9% of the cases as secondary dengue infections, respectively. Cohen's kappa coefficient and McNemar's association tests demonstrated a significant disagreement between the two diagnostic strategies and ELISPOT-MNT. Overall, these results evidence the relatively poor performances of commercial assays for the diagnosis of acute and secondary dengue infections, compared with ELISPOT-MNT, and raise concern about the accuracy of these assays for the diagnostic of dengue in endemic areas.

Multicountry prospective clinical evaluation of two enzyme-linked immunosorbent assays and two rapid diagnostic tests for diagnosing dengue fever.

We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks.

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

BackgroundEarly and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.MethodsA single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).ResultsAcute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.ConclusionThe RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

Altered Enzyme-Linked Immunosorbent Assay Immunoglobulin M (IgM)/IgG Optical Density Ratios Can Correctly Classify All Primary or Secondary Dengue Virus Infections 1 Day after the Onset of Symptoms, when All of the Viruses Can Be Isolated

We compared dengue virus (DV) isolation rates and tested whether acute primary (P) and acute/probable acute secondary (S/PS) DV infections could be correctly classified serologically when the patients' first serum (S1) samples were obtained 1 to 3 days after the onset of symptoms (AOS). DV envelope/membrane protein-specific immunoglobulin M (IgM) capture and IgG capture enzyme-linked immunosorbent assay (ELISA) titrations (1/log(10) 1.7 to 1 log(10) 6.6 dilutions) were performed on 100 paired S1 and S2 samples from suspected DV infections. The serologically confirmed S/PS infections were divided into six subgroups based on their different IgM and IgG responses. Because of their much greater dynamic ranges, IgG/IgM ELISA titer ratios were more accurate and reliable than IgM/IgG optical density (OD) ratios recorded at a single cutoff dilution for discriminating between P and S/PS infections. However, 62% of these patients' S1 samples were DV IgM and IgG titer negative (<OD(max)/2 titer threshold), and in 35% of the S/PS infections, the patients' S1 and S2 samples were IgM titer negative. The IgM OD values were, however, much higher than those of IgG in the S1 samples of many of these, and the other, S/PS infections. This necessitated using higher (>or=2.60 and <2.60) discriminatory IgM/IgG OD (DOD) ratios on these S1 samples than those published previously to correctly classify the highest percentage of these P and S/PS infections. The DV isolation rate was highest (12/12; 100%) using IgG and IgM titer-negative S1 samples collected 1 day AOS, when 100% of them were correctly classified as P or S/PS infections using these higher DOD ratios.

Detection of specific antibodies in saliva during dengue infection.

Saliva was collected prospectively from patients presenting with suspected dengue infection 4 to 8 days after the onset of symptoms and assayed by a commercial dengue immunoglobulin M (IgM) and IgG capture enzyme-linked immunosorbent assay (ELISA) (PanBio Dengue Duo ELISA). Laboratory diagnosis was based on virus isolation and on hemagglutination inhibition (HAI) assay and an in-house IgM and IgG capture ELISA. With a positive result defined as either salivary IgM or IgG levels above the cutoff value, an overall sensitivity of 92% was obtained for both primary- and secondary-dengue patients (22 of 24), while no patients with non-flavivirus infections (n = 11) and no healthy laboratory donors (n = 17) showed elevation of salivary antidengue antibody (100% specificity). Salivary IgG levels correlated well with serum HAI titer (r = 0.78), and salivary IgG levels could be used to distinguish between primary- and secondary-dengue virus infections.

Novel assay for the detection of immunoglobulin G antihuman immunodeficiency virus in untreated saliva and urine.

Epidemiological evidence and laboratory studies indicate that human immunodeficiency virus type 1 (HIV 1) is rarely, if ever, transmitted in saliva or urine. In that both specimens are easy to collect, each may be a useful alternative to serum specimens for anti-HIV screening. A rapid, simple, and robust IgG-capture enzyme-linked immunosorbent assay (GACELISA) suitable for the detection of anti-HIV 1 and 2 in saliva and urine was developed. Following optimisation of the assay, 177 salivary and 568 urine specimens collected from individuals of known serostatus were investigated. The assay was 100% sensitive on 50 salivary (median OD/CO = 8.9) and 126 urinary (median OD/CO = 8.6) specimens collected from anti-HIV-positive patients. The specificity was 100% on 127 salivary specimens (median OD/CO = 0.37) and 422 urinary specimens (median OD/CO = 0.39) collected from anti-HIV-negative individuals. These findings demonstrate that GACELISA HIV 1 + 2 tests on saliva or on urine are an accurate alternative to a conventional anti-HIV test of blood. This assay is satisfactory for surveillance purposes and, with appropriate precautions, could be used clinically.

Intrathecally produced antibodies to Borrelia burgdorferi measured by IgG-capture ELISA

An IgG-capture enzyme-linked immunosorbent assay (ELISA) which directly measures the intrathecal production of Borrelia burgdorferi-specific IgG in neuroborreliosis is described. The method is based on the simultaneous measurement of IgG-specific antibodies in paired cerebrospinal fluid (CSF) and serum samples. Sixty-nine paired CSF/serum samples from 64 patients with neuroborreliosis or other neurological disorders and six samples from acrodermatitis chronica atrophicans (ACA) patients were analysed by IgG-capture ELISA. Of 12 samples from patients with early neuroborreliosis, intrathecal production of B. burgdorferi-specific IgG could be demonstrated in 11 samples. Of 56 patients with other neurological disorders of known or unknown etiology, intrathecal production of B. burgdorferi-specific IgG was demonstrated in one patient who had a history compatible with a previous neuroborreliosis. No intrathecal production was found in the samples from ACA patients in spite of high Borrelia titres in their CSF as measured by indirect ELISA. In conclusion, the B. burgdorferi IgG-capture ELISA is a sensitive and reliable method for the detection of intrathecal production of IgG antibodies to B. burgdorferi. It is advantageous in comparison to the indirect ELISA as it does not require correction for leakage across the blood/CSF barrier.

Serodignosis of herpes simplex encephalitis by antibody capture enzyme-linked immunosorbent assay

The sensitivity of a herpes simplex virus (HSV) IgG capture enzyme-linked immunosorbent assay was tested in 103 cerebrospinal fluid (CSF) and serum samples from 47 patients with herpes simplex encephalitis (HSE). Significant differences between CSF and serum absorbances were found in all HSE patients; earlier in three out of 30 patients by the capture technique than by the indirect ELISA. In follow-up samples with exceedingly high anti-HSV activity, maximal absorbance values were found in CSF and serum resulting in falsely-negative absorbance differences. Reanalysis with a lower concentration of capture anti-IgG solved the problem. This lower anti-IgG concentration was found suboptimal for analysis of early specimens. The specificity was tested in specimens from 115 patients without intrathecal anti-HSV production as determined by indirect ELISA. Five out of 73 patients with focal encephalitis of non-HSV origin had low level positive capture results. Four of them had varicella zoster (VZ) meningoencephalitis. Severe blood-CSF barrier damage did not interfere in seven patients tested. The IgG-capture was found advantageous to the indirect technique as the results were more sensitive and clearcut. It is less labour intensive and may be completed within five to six hours.