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.

Development of a Human-Murine Chimeric Immunoglobulin M for Use in the Serological Detection of Human Alphavirus Antibodies

“…We previously reported on the construction and utility of human-murine chimeric monoclonal antibodies (cMAbs) derived from group-specific murine MAbs (mMAbs) as substitutes for antibody-positive human control sera in the serodiagnosis of human alphaviral and flaviviral infections. The flavivirus group-specific mMAb 6B6C-1 was used to develop an IgG cMAb (6GF4) and an IgM cMAb (6ME2) that were successfully employed as positive controls in the flavivirus indirect IgG ELISA and MAC-ELISA, respectively (4,5). A similar IgG cMAb (1GD5) was constructed using the alphavirus group-specific mMAb 1A4B-6 for use in the serodiagnosis of human alphaviral disease (5).…”

“…The isolation, sequencing, and cloning of the 1A4B-6 mMAb heavy and kappa V regions (V H and V K ) have been previously described; the sequences for these regions can also be accessed via GenBank using the following accession numbers: 1A4B-6 V K , GU724342; 1A4B-6 V H , GU724341 (4). During the development of the 1GD5 IgG cMAb, the 1A4B-6 V H and V K regions were modified by PCR to incorporate partial 5Ј leader sequences, 3Ј splice donor junctions, and appropriate restriction endonuclease sites for subsequent ligation with the Abbott human IgG expression vector pdHL2 (4).…”

“…During the development of the 1GD5 IgG cMAb, the 1A4B-6 V H and V K regions were modified by PCR to incorporate partial 5Ј leader sequences, 3Ј splice donor junctions, and appropriate restriction endonuclease sites for subsequent ligation with the Abbott human IgG expression vector pdHL2 (4). These modifications also allowed the 1A4B-6 V regions to be incorporated into the Abbott human IgM expression vector pJH2, forming plasmid pJH-1M, which was subsequently used to transform murine Sp2/0-AG14 (Sp2) cells by electroporation as previously described (4,5). Sp2 cells neither secrete nor synthesize murine IgG.…”

“…The clone (1MD11) exhibiting the strongest reaction with the EEEV SMB antigen was expanded and used in subsequent studies to determine alphavirus group specificity. The details concerning the screening of transfected cells by ELISA have been described previously (4,5).…”

“…We have previously reported on the development of an IgM cMAb, 6ME2, for use in diagnosis of flavivirus infections by MAC-ELISA as well as IgG cMAbs 1GD5 and 6GF4 for use in either the alphavirus or flavivirus (respectively) indirect IgG ELISA (4,5). The addition of the alphavirus group-specific 1MD11 IgM cMAb to this collection completes the set of cMAbs necessary to identify both classes of human antibodies that develop in a wide variety of human arboviral infections.…”