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Development of human-murine chimeric immunoglobulin G for use in the serological detection of human flavivirus and alphavirus antibodies.

ABSTRACT: Diagnosis of human arboviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the indirect IgG ELISA. Broad application of these assays is hindered by the lack of standardized positive human control sera that react with a wide variety of flaviviruses (e.g., dengue, West Nile, yellow fever, Japanese encephalitis, Saint Louis encephalitis, and Powassan viruses), or alphaviruses (e.g., Eastern equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis, and chikungunya viruses) that can cause human disease. We have created human-murine chimeric monoclonal antibodies (cMAbs) by combining the variable regions of flavivirus (6B6C-1) or alphavirus (1A4B-6) broadly cross-reactive murine MAbs (mMAbs) with the constant region of human IgG1. These cMAbs may be used as standardized reagents capable of replacing human infection-immune-positive control sera in indirect IgG ELISA for diagnosis of all human flaviviral or alphaviral infections. The IgG cMAbs secreted from plasmid-transformed Sp2/0-Ag14 cells had serological activity identical to that of the parent mMAbs, as measured by ELISA using multiple flaviviruses or alphaviruses.

SUBMITTER: Thibodeaux BA

PROVIDER: S-EPMC2952999 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Development of human-murine chimeric immunoglobulin G for use in the serological detection of human flavivirus and alphavirus antibodies.

Thibodeaux Brett A BA Panella Amanda N AN Roehrig John T JT

Clinical and vaccine immunology : CVI 20100825 10

Diagnosis of human arboviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the indirect IgG ELISA. Broad application of these assays is hindered by the lack of standardized positive human control sera that react with a wide variety of flaviviruses (e.g., dengue, West Nile, yellow fever, Japanese encephalitis, Saint Louis encephalitis, and Powassan viruses), or alphaviruses (e.g., Eastern e ...[more]

PMID: 20739503

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Project description:BackgroundHuman cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity.MethodsHere we investigated whether luciferase immunoprecipitation systems (LIPS) would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as Renilla luciferase (Ruc) fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA.ResultsAnalysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100-1000 fold higher in the CMV positive sera compared to the CMV negative samples (p < 0.0001) and infection status exactly matched the ELISA results for the 46 samples of the first cohort (100% sensitivity and 100% specificity). Two additional antigen fragments, pp65-d1 and pp65-d2 also showed robust antibody titers in some CMV-infected sera and yielded 50% and 96% sensitivity, respectively. Analysis of a second cohort of 70 samples using a mixture of the 4 antigens, which simplifies data collection and analysis, yielded values which correlated well with the sum of the values from the 4 separate tests (rs = 0.93, p < 0.00001). While comparison of the LIPS results from this second cohort with ELISA showed 100% sensitivity, LIPS detected six additional CMV positive samples that were not detected by ELISA. Heat map analysis revealed that several of the LIPS positive/ELISA negative samples had positive LIPS immunoreactivity with 3-4 of the CMV antigens.ConclusionThese results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.

Dataset Information

Development of human-murine chimeric immunoglobulin G for use in the serological detection of human flavivirus and alphavirus antibodies.

Publications

Development of human-murine chimeric immunoglobulin G for use in the serological detection of human flavivirus and alphavirus antibodies.

Similar Datasets

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