Comparative Analysis of Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay Using Virus-Like Particles or Virus-Infected Mouse Brain Antigens To Detect IgM Antibody in Sera from Patients with Evident Flaviviral Infections

Abstract

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquitoborne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 ؊ specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain. Members of the genusFlavivirus have an ϳ11-kb, singlestranded, positive-sense RNA genome which translates and encodes capsid (C), premembrane/membrane (prM/M), and envelope (E) structural proteins and seven nonstructural proteins. During natural flavivirus infection, noninfectious viruslike particles (VLPs) are produced in addition to infectious, mature virions (25). Flavivirus VLPs have structural and physiochemical properties similar to mature virus particles. VLPs have been characterized ...

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

“…Although VLP-MAC-ELISA and InBios DENV Detect IgM capture ELISA detected anti-prM/E antibodies, the different sensitivities of the two assays could be due to the determination of the cutoff value used in each assay. In-house VLP-MAC-ELISA calculates the cutoff value by dividing the OD values from the testing serum and the negative serum control using VLPs as the antigens (18). The InBios DENV Detect IgM capture ELISA determines the immune status ratio (IMR) by dividing the OD value of the testing serum using VLPs with the OD value of testing serum using negative antigens (as shown in the product insert of the InBios kit).…”

“…Molecular cloning procedures to construct these plasmids were described previously (15,16). The VLPs and soluble NS1 protein (sNS1) were expressed by COS-1 cells electroporated with recombinant expression plasmids containing the prM/E and NS1 genes, respectively, using the protocol described previously (17,18). Electroporated cells were recovered in 50 ml DMEM, seeded into three separate 75-cm 2 culture flasks for VLP/sNS1 expression, and incubated at 28°C with 5% CO 2 .…”

“…Antigen-capture ELISAs (Ag-ELISAs) were used to detect and quantify secreted antigens from the prM/E and NS1 gene-carrying-plasmid-transformed COS-1 cells as described previously (17,18). Anti-DENV-1 to -4, WNV, or JEV VLP and NS1 polyclonal rabbit sera were used to capture the antigens.…”

“…Human sera were assayed for the presence of prM/E-and sNS1-specific antibodies using both MAC-and GAC-ELISAs as previously described (18). Briefly, 96-well plates were coated with goat anti-human IgM or IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) that was diluted 1:2,000 in phosphate-buffered saline (PBS) buffer and incubated overnight at 4°C.…”

Evaluation of IgM Antibody Capture Enzyme-Linked Immunosorbent Assay Kits for Detection of IgM against Japanese Encephalitis Virus in Cerebrospinal Fluid Samples

“…The DENV and JEV antigens from stably transformed COS-1 cells that secrete JE-or DEN virus-like particles into the supernatant were used in the CDC MACELISAs. [14][15][16][17] Using tetramethylbenzidine as the substrate, we determined the positive-to-negative absorbance ratios (P/Ns) by dividing the absorbance at 450 nm (A 450 ) of the CSF specimen tested with positive antigen by the average of the A 450 of the normal control CSF tested with viral antigen. A P/N ≥ 3 was considered positive, ≥ 2 but < 3 equivocal, and < 2 negative.…”