Antibody-positive human control sera for use in the MAC and indirect IgG ELISA are derived from small volumes of serum specimens submitted to the CDC for diagnostic purposes. Not surprisingly, these specimens are typically collected only from the most prevalent arboviral infections, thus limiting the application of ELISA in arboviral surveillance to the narrow range of diseases currently being diagnosed or tracked by disease surveillance systems at any given time. Furthermore, positive control serum pools prepared from these specimens suffer high lot-to-lot variability that necessitates constant recalibration of serological potency and coverage. Of even greater concern is the lack of antibody-positive control sera that can be used in these ELISAs for the identification of infrequent or emerging arboviruses (20
). In this study, we have demonstrated that IgG cMAbs expressing the variable region specificity of the mMAbs 6B6C-1 (cMAb 6GF4) or 1A4B-6 (cMAb 1GD5) can serve as suitable replacements for human control sera in the indirect IgG ELISA used in the serodiagnosis of arboviral disease. These cMAbs offer diagnostic laboratories an unlimited supply of control reagents of a set affinity and specificity, in known quantities that should facilitate diagnostic testing and lab-to-lab comparative evaluations.
A number of techniques have recently been described for engineering human antibodies. Transgenic mouse strains carrying human heavy- and light-chain loci, the immortalization of human B cells through viral transformation, and production of human hybridomas using new human fusion partner cell lines are all methods capable of producing human monoclonal antibodies (6
). Unfortunately, these methods do not facilitate the design of human MAbs of a defined specificity. A considerable amount of additional screening would be required to identify specific group-reactive antibodies. An alternative to producing fully human MAbs is humanizing existing murine MAbs of known specificity. The flavivirus group-specific 6B6C-1 mMAb was originally raised against SLEV and is specific for the flaviviral envelope (E) protein (22
); mMAb 1A4B-6 reacts specifically with the E1d domain of the EEEV E1 glycoprotein and is alphavirus group reactive (14
). Both 6B6C-1 and 1A4B-6 are regularly used in serological assays as capture antibodies and antibody-enzyme conjugate detectors and were likely candidates for humanization (15
). Using the pdHL2 IgG expression vector along with RNA purified from the 6B6C-1 and 1A4B-6 hybridomas, we prepared IgG cMAbs 6GF4 and 1GD5 for use in the indirect IgG ELISA.
The 6GF4 IgG cMAb was able to achieve positive P/N values with each flaviviral antigen tested in the indirect IgG ELISA. 6GF4 demonstrated a strong preference for the YFV, WNV, and JEV antigens compared to the relatively weaker reactions with DENV-2, POWV, or SLEV antigens. The 1GD5 IgG cMAb similarly reacted positively with all alphaviruses tested, and there appeared to be much less variability in the reactivity of 1GD5 when assayed against the representative members of the alphavirus family.
Although it was somewhat surprising to see that the 6GF4 cMAb reacted weakly with SLEV, the virus initially used to generate the 6B6C-1 mMAb, it is not all that alarming considering that the reactivity and quality of SMB and VLP antigens can vary greatly from one lot of antigen to the next. Furthermore, when tested against viral seed antigens instead of the SMB or VLP antigens in the indirect IgG ELISA format, the 6GF4 cMAb reacted better with the SLEV seed antigen, generating the second-highest overall Pmax
/N value (4.65) of all flavivirus viral seed antigens tested. The 6ME2 IgM cMAb described in a previous publication also demonstrated a relatively low reactivity with the SLEV SMB antigen and reacted strongly with the SLE viral seed antigen (32
). This disparity between the viral SMB antigen and the viral seed antigen might be attributed to differences inherent in the SLEV strains used and/or differences in the preparation of each antigen; the SLEV SMB antigen used in both the IgG and IgM ELISA studies was prepared from strain TBH-28, an isolate taken postmortem from cases of fatal SLE in the Tampa Bay area of Florida in 1962, while the viral seed antigen was prepared from Vero cells infected with SLEV strain MSI-7, the same SLE strain that was originally used to generate the 6B6C-1 mMAb. However, given the homogeneity of the 6B6C-1 epitope among all flaviviruses (5
), the variable reactivity of 6GF4 with different flaviviral antigens is most likely due to the quality and concentration of the specific antigen lots available from the CDC DRL used in this assay rather than the preference of the chimeric antibody for a specific flavivirus.
The viral antigens included in these ELISAs are representative of the major flavivirus or alphavirus antigen complexes with members causing human disease. Therefore, it is reasonable to assume that these chimeric antibodies would be useful as positive controls in the serodiagnosis of all flaviviral or alphaviral specimens. One possible complication with using a MAb-derived chimeric antibody as a positive control reagent would be the occurrence of an arbovirus with an altered E glycoprotein (flavivirus) or E1 glycoprotein (alphavirus) rendering it nonreactive with the group-specific activities of 6B6C-1 or 1A4B-6. The epitope defined by 6B6C-1 and other E protein-specific flavivirus group-reactive mMAbs has recently been mapped to the E protein fusion loop (5
). This sequence is highly conserved among all flaviviruses, probably because of its critical interaction with cell membranes during virus replication. The alphavirus E1 glycoprotein is also reported to be responsible for membrane fusion, and although no epitope map yet exists for the alphavirus E1 glycoprotein, the crystal structure of E1 has been determined and was found to share significant homology and identical topology with the flavivirus E glycoprotein (7
). Furthermore, we have shown that a flavivirus transmitted by a vector other than the mosquito, POWV, reacts with the 6GF4 IgG cMAb. This finding agrees with those previously reported by the CDC DRL that both tick-borne encephalitis virus and POWV react with the 6B6C-1 mMAb in the MAC-ELISA format (32
Given the respective group reactivities of 6B6C-1 and 1A4B-6, the discovery of a nonreactive flavivirus or alphavirus is unlikely. If such a virus was isolated, however, one solution would be to develop a combination of two different chimeric antibodies sharing group reactivity but for separate epitopes. Additionally, since the current indirect IgG-ELISA formats utilize a 6B6C-1-enzyme conjugate detector in flavivirus serodiagnosis and a 1A4B-6 capture antibody in alphaviral serodiagnosis, any arbovirus expressing a mutated or nonreactive glycoprotein would not likely be detected by the currently employed assays.
The incorporation of cMAbs in immunoassays that rely on variably reactive human sera as controls will provide diagnostic laboratories with an unlimited supply of control reagents of a set affinity and specificity. Also, the use of a positive control with the specificity of a MAb allows for better characterization of unknown specimens than that of polyclonal sera with heterogeneous reactivities. The 6GF4 and 1GD5 IgG cMAbs should offer viable alternatives to positive human serum controls for flavivirus or alphavirus detection via the indirect IgG ELISA. Together with the previously described 6ME2 IgM cMAb, we now possess positive controls capable of evaluating both acute- and convalescent-phase sera for possible flavivirus infection. An IgM cMAb with alphavirus group specificity has also been developed, is currently being characterized for utilization in the MAC-ELISA for serodiagnosis of alphaviral disease, and will complete the set of cMAbs necessary to identify both classes of human antibodies developed in a wide variety of human arboviral infections.