Establishment of a Competitive Binding Assay Identifying the Different Characteristics of Neutralizing Epitopes of...

To confirm the different characteristics of genotype-specific and common neutralizing epitopes of hepatitis E virus (HEV).

A competitive binding assay was established with known genotype-common neutralizing monoclonal antibodies (mAbs) 3G1 and 5G5 as well as genotype-specific neutralizing mAbs 2B1 and 4C5. HEV ORF2 recombinant p166W01 derived from genotype 1 and p166Chn derived from genotype 4 were used as coated antigens, to determine whether the mAbs recognize independent, similar, or overlapping epitopes. mAbs were produced, purified, and conjugated with horseradish peroxidase (HRP). HRP-conjugated 2B1 could react only with p166W01 but not p166Chn, HRP-conjugated 4C5 could react only with p166Chn but not p166W01, while HRP-conjugated 3G1 and 5G5 could react both with p166W01 and p166Chn. Thus, competitive binding assays were performed successively using p166W01 and p166Chn antigen.

The results of competitive binding assays revealed that the binding of HRP-conjugated 2B1 to p166W01 could not be inhibited by 5G5 or 3G1. Similarly, the binding of HRP-conjugated 4C5 to p166Chn could not be inhibited by 5G5 or 3G1. However, the mAbs 5G5 and 3G1 blocked each other's binding to p166W01 and p166Chn, suggesting that common and genotype-specific neutralizing mAbs recognize independent epitopes.