Development of HEK-293 Cell Lines Constitutively Expressing Flaviviral Antigens for Use in Diagnostics

Powers, Jordan A.; Skinner, Benjamin; Davis, Brent S.; Biggerstaff, Brad J.; Robb, Lucy; Gordón, Elizabeth; Calvert, Amanda E.; Chang, Gwong‐Jen J.

doi:10.1128/spectrum.00592-22

Cited by 4 publications

(7 citation statements)

References 66 publications

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“…HEK-293 cells were electroporated with expression vectors containing the human-murine chimeric IgM constructs as previously described ( 59 ). Briefly, 30 µg of DNA per reaction was added to 0.5 mL of cells at a cell density of 1–2 × 10 7 cells/mL.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were recovered overnight in growth medium and reseeded the next day at 1 × 10 6 cells/mL. Immunofluorescence assays (IFAs) were performed after recovery to verify the expression of the gene construct as previously described using a goat-anti-human IgM Fc 5µ antibody conjugated to fluorescein (Cat#109-095-043, Jackson Immunoresearch, West Grove, PA, USA) ( 59 ). The process for the selection of human-murine immunoglobulin expressing HEK-293 cells was adapted from previous methodology used to develop HEK-293 cell lines expressing flavivirus diagnostic antigens using 3% carboxymethyl cellulose in growth media ( 59 ).…”

Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence assays (IFAs) were performed after recovery to verify the expression of the gene construct as previously described using a goat-anti-human IgM Fc 5µ antibody conjugated to fluorescein (Cat#109-095-043, Jackson Immunoresearch, West Grove, PA, USA) ( 59 ). The process for the selection of human-murine immunoglobulin expressing HEK-293 cells was adapted from previous methodology used to develop HEK-293 cell lines expressing flavivirus diagnostic antigens using 3% carboxymethyl cellulose in growth media ( 59 ). Cells were grown for 7–14 days, and colonies were picked using the ClonePix2 (Molecular Devices, Sunnyvale, CA, USA) and placed into 96-well plates (Corning, Tewksbury, MA, USA) containing growth media.…”

Section: Methodsmentioning

confidence: 99%

“…CSGV hIgM stable expression from HEK-293 cells was determined as previously described ( 59 ). Cells were passaged 10 times, and cell-culture supernatants from each passage were tested in the MAC-ELISA in duplicate.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were passaged 10 times, and cell-culture supernatants from each passage were tested in the MAC-ELISA in duplicate. Regression spline fits of expression ( P / N ratio) against log-dilution were fitted to all passages and a mean curve was estimated using methods of Heckman et al ( 60 ) and Powers et al ( 59 ). The endpoint titer (EPT) was estimated from the spline curves for each passage as the lowest dilution that resulted in a positive ( P / N ) ratio of 3.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics

Powers,

Boroughs,

Mikula

et al. 2023

Microbiol Spectr

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California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA. IMPORTANCE Orthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%