The characterization of four anti-Ft LPS IgG2a MAbs in the current study showed that all bind to OAg, with FB11 binding to a terminal epitope, and Ab3, Ab52, and Ab54 binding to internal epitopes. Binding of FB11 to a terminal epitope of OAg in contrast to the binding of the other three MAbs to internal OAg epitopes is supported by three independent experiments: (1) Western blot analysis on Ft LVS, showing that FB11 binds with equal intensity to both long and short LPS chains whereas binding of the other three MAbs decreases with decreasing LPS chain length; (2) ELISA on plates coated with decreasing concentrations of LPS, showing that the binding of FB11 decreases much quicker than the binding of the other three MAbs with decreasing LPS concentrations; and (3) the linear carbohydrate immunoprecipitation assay showing the inability of FB11, but not of the other three MAbs, to immunoprecipitate Ft OAg despite the superiority of FB11 in agglutinating LVS bacteria.
The linear carbohydrate immunoprecipitation assay was first used by Cisar and colleagues(40)
to distinguish between end-binding and internal-binding anti-carbohydrate MAbs. In the original assay, an end-binding anti-dextran myeloma antibody was distinguished from an internal-binding anti-dextran myeloma antibody by the inability of the end-binding antibody to immunoprecipitate a linear α(1
6)-linked dextran (which contains a single non-reducing end per molecule) despite the ability of both antibodies to immunoprecipitate a branched dextran (95% α(1
3), which contains multiple non-reducing ends per molecule). Based on this assay, Cisar and colleagues(40)
suggested that the antibody binding to the terminal epitope has a “cavity-like” binding site whereas the antibody binding to a non-terminal (internal) epitope has a “groove-like” binding site. Cavity-like and groove-type sites for anti-carbohydrate antibodies, including antibodies to LPS OAg from Shigella flexneri
, Vibrio cholerae
, and Brucella abortus
, were supported by immunochemical, computer modeling, and x-ray crystallographic studies.(41–50)
These showed a maximum of five sugar residues accommodated by cavity-type sites and six to eight sugar residues accommodated by groove-type sites. In the current study, FB11 may have a cavity-type site and the other three anti-Ft OAg MAbs likely have groove-type sites. Consistent with a cavity-type (or pocket-like) site, which buries more of the epitope and is therefore expected to have higher complementarity, and hence affinity and avidity, for Ag than groove-type sites,(40)
the avidity of FB11 for OAg is higher than those of Ab3, Ab52, and Ab54, as suggested by ELISA and Western blot analysis and demonstrated by Biacore analysis. The affinity of FB11 (4.01
M), determined by Biacore analysis, is in the upper range of affinities reported for MAbs specific for the OAg of Shigella flexneri
M) and Vibrio cholerae
M) or for α(1
6) dextran (1.5
The affinity of Ab52 could not be determined by Biacore analysis of immobilized MAb binding to soluble Ft OAg because of the multivalent binding of immobilized Ab52 to each soluble OAg molecule. Instead, the equilibrium dissociation constant for this multivalent interaction was calculated to be 4.37
M, which describes an essentially irreversible reaction. This very tight “Velcro-like” interaction of soluble OAg with immobilized Ab52 illustrates the in vivo
advantage of B cells expressing (immobilized) internal-binding surface B cell receptors at holding onto LPS and becoming activated. The prevalence of internal-binding anti-Ft LPS antibodies is therefore not surprising: although FB11 was reported to have been obtained from BALB/c mice immunized with LVS or with Ft LPS in complete Freund's adjuvant, using “different immunization schemes,”(33)
no other anti-Ft LPS MAb, obtained by us or described by others, shows binding to the short chains of the LPS ladder in Western blot analyses.(31,53–56)
This suggests that the repeating internal epitopes of Ft-LPS OAg are much more immunogenic than the OAg ends, even though, evidently, BALB/c mice have the ability to produce FB11-like antibodies.
Thus, the current findings demonstrate that antibodies to non-overlapping epitopes of Ft OAg can be generated and suggest that antibodies targeting terminal epitopes of Ft OAg are more difficult to induce but may have higher avidities than those targeting internal OAg epitopes. End-binding antibodies may therefore be beneficial in defense against Ft. Because end-binding anti-Ft LPS antibodies are distinguished from internal-binding anti-LPS antibodies by their ability to bind to short LPS chains, our results support the use of short-chain LPS or short-chain OAg as a tularemia vaccine component.