MAPPING OF EPITOPES FOR CITRUS TRISTEZA VIRUS-SPECIFIC MONOCLONAL-ANTIBODIES USING BACTERIALLY EXPRESSED COAT PROTEIN-FRAGMENTS

Epitopes for a panel of 30 monoclonal antibodies (MAbs) specific for c itrus tristeza virus (CTV) were mapped on the CTV coat protein (CP) ex pressed in bacterial cells. Expression constructs that generated diffe rent portions of the CTV CP were screened against MAbs by Western blot ting and enzyme-linked immunosorbent assay. All MAbs analyzed could be placed into five groups (I to V), four of which have continuous seque ntial epitopes. Group I has an epitope within the nine C-terminal amin o acids (aa), 215 to 223, that reacted only to MAb 4H6; the group II e pitope was mapped between aa 173 and 215 and reacted to MCA-14; the gr oup III epitope was mapped between aa 118 and 128 and reacted to five MAbs, including MCA-13; and the group IV epitope was mapped between aa 2 and 121 and reacted to four MAbs. Epitope(s) for a large group of M Abs (group V) either were conformational or included a conformational element, because they only reacted with the complete CP fusion protein and not with its fragments. Specific proteolytic cleavage of a CP fus ion protein expressed in Escherichia coli as a peptide fused to a malt ose-binding protein (MBP) released a full-size CP with essentially no reactivity with MAbs from group V. Additional studies will be needed t o differentiate members of this group. The linear, continuous epitope for MCA-13 (aa 118 to 128), which distinguishes Florida quick decline- inducing (D-I) CTV isolates from mild isolates, was expressed in E. co li cells as a peptide fused to an MBP. This fusion protein was purifie d and used as antigen to generate a rabbit polyclonal antiserum. The a a 118 to 128-specific polyclonal antiserum had the same serological pr operties as MAb MCA-13 and reacted with Florida quick D-I CTV isolates but not with mild isolates.