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Clin Vaccine Immunol. 2011 December; 18(12): 2181–2182.
PMCID: PMC3232697

Development of a Human-Murine Chimeric Immunoglobulin M for Use in the Serological Detection of Human Alphavirus Antibodies [down-pointing small open triangle]


Diagnosis of human alphaviral infections relies on serological techniques, such as the immunoglobulin M antibody capture–enzyme-linked immunosorbent assay (MAC-ELISA). We have humanized the alphavirus broadly cross-reactive murine monoclonal antibody 1A4B-6 to create a reagent capable of replacing human positive sera in the MAC-ELISA for diagnosis of human alphaviral infections.


Arthropod-borne viruses (arboviruses) are positive-stranded RNA viruses responsible for a number of medically important human diseases. Although over 150 arboviruses are known to cause disease in humans, the majority of medically important arboviruses are found in three separate families, the Flaviviridae, the Togaviridae (genus Alphavirus), and Bunyaviridae (2). Alphaviruses remain important emerging mosquito-borne, zoonotic pathogens that cause both localized human outbreaks and epizootic (e.g., Venezuelan equine encephalitis) and large human (e.g., chikungunya) epidemics. Alphaviruses can be divided into seven serocomplexes, four of which, represented by Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), and Semliki Forest virus, contain most of the medically important alphaviruses (1). Rapid serologic assays such as the immunoglobulin M (IgM) antibody capture–enzyme-linked immunosorbent assay (MAC-ELISA) and IgG ELISA are now routinely used in diagnosis soon after infection, usually 8 to 45 days after onset of symptoms (1). In many cases, a positive MAC-ELISA with an acute-phase serum sample precludes the need for testing of a convalescent-phase serum sample.

Application of the ELISA in serodiagnosis of arboviral infection is most hampered by the limited availability of human infection-immune sera for use as virus-reactive, antibody-positive control specimens. 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). In this report we describe the development and characterization of a new IgM cMAb for use in the alphavirus MAC-ELISA. This cMAb (1MD11) was created by incorporating the variable (V) regions of 1A4B-6 into a plasmid construct containing the human Ig μ chain. The alphaviral group reactivity of 1MD11 was evaluated by MAC-ELISA using representatives from each of the four medically important alphavirus serocomplexes.

The isolation, sequencing, and cloning of the 1A4B-6 mMAb heavy and kappa V regions (VH and VK) have been previously described; the sequences for these regions can also be accessed via GenBank using the following accession numbers: 1A4B-6 VK, GU724342; 1A4B-6 VH, GU724341 (4). During the development of the 1GD5 IgG cMAb, the 1A4B-6 VH and VK regions were modified by PCR to incorporate partial 5′ leader sequences, 3′ 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. Sp2 cells transfected with the pJH-1M plasmid were screened for human IgM production by ELISA; cells demonstrating human IgM in supernatants were next screened for antialphavirus reactivity by ELISA using EEEV (strain NJ/60) suckling mouse brain (SMB) antigen. 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).

Quantitative analysis of the 1MD11 supernatant indicated an IgM cMAb concentration of 0.25 μg/ml, approximately 0.0025% of the total protein content of the supernatant. The 1MD11 supernatant was next assayed for alphavirus-specific reactivity by MAC-ELISA using SMB antigens representing the four medically important alphavirus serocomplexes. SMB antigens for EEEV (strain NJ/60), VEEV (strain TC83), WEEV (strain McMillan), and chikungunya virus (CHIKV; strain S27), along with IgM-positive human control sera for EEEV, VEEV, and CHIKV, were provided by the CDC Diagnostic and Reference Laboratory (DRL; Fort Collins, CO). Use of the MAC-ELISA for detection of arbovirus-specific human-murine cMAbs has been previously described (5); IgM-positive human control sera for WEEV were not available. Test validation and positive-to-negative ratio (P/N) values were determined as follows. The N value for a given viral antigen was defined as the average A450 value of negative human serum (NHS; diluted 1:400) when reacted with that antigen. The positive (P) value for a given viral antigen was determined to be the average A450 value of 1MD11 IgM cMAb or positive human serum (PHS) when reacted with that antigen. The Pmax value for a given antigen and 1MD11 IgM cMAb was defined as the maximum P value observed in a dilution series of 1MD11 IgM cMAb reacted with that antigen; for a given antigen and PHS, the Pmax value was defined as the P value of PHS at the dilution recommended by the CDC DRL (1:400). Dilutions of 1:16 or greater of the 1MD11 supernatant were able to yield positive ELISA reactions (P/N, >3.0) with all alphaviral antigens assayed (Table 1). The 1MD11 IgM cMAb demonstrated higher Pmax values against the VEEV and CHIKV than to EEEV and WEEV, which is somewhat surprising when one considers the fact that the 1MD11 IgM cMAb utilizes the variable regions of mMAb 1A4B-6, which was initially developed against EEEV strain NJ/60 (3). However, given the lot-to-lot variability inherent in SMB viral antigen production, the differences in Pmax values observed are most likely attributable to differences in viral antigen quality as opposed to increased affinity of the 1MD11 IgM cMAb to VEEV or CHIKV.

Table 1.
Reactivities of the 1MD11 IgM cMAb with alphavirus SMB antigens in a MAC-ELISA

The use of cMAbs in arboviral immunoassays provides diagnostic laboratories with a favorable alternative to the use of variably reactive human sera commonly used as positive and negative controls. 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.


This work was supported, in part, by a postdoctoral fellowship awarded to B.A.T. by the American Society for Microbiology and the Coordinating Center for Infectious Diseases.


[down-pointing small open triangle]Published ahead of print on 5 October 2011.


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