Epitope size, specificity and equilibrium constant for four monoclonal antibodies binding to the O:4 polysaccharide antigen of Salmonella serogroup B bacteria.

One rat (MAST 83) and three mouse (MAST 107, 108 and 112) monoclonal antibodies (mAbs) directed against Salmonella serogroup BO lipopolysaccharide (LPS) were characterized and found to bind to the O:4 epitope but recognizing different surfaces of the polysaccharide chain. The epitopes were defined from the combined results of: (i) binding specificities in enzyme immuno assay (EIA) against chemically defined LPS and glycoconjugates; (ii) studies of affinity constants in Farr-assay for binding to oligosaccharides purified from LPS, or chemically synthesized; and (iii) knowledge of the conformation of the O-polysaccharide chain of Salmonella BO bacteria. Two of the antibodies, MAST 83 and 108, bound to the O:4 epitope when present in the terminal non-reducing end as well as an intrachain determinant of the O-polysaccharide, whereas MAST 107 and 112 bound only to the O:4 epitope when present as an intrachain determinant. The equilibrium constants (K values), determined for binding of the mAbs and a Fab-fragment isolated from one of them to a 125l-labelled tyramine-derivative of a Salmonella BO dodecasaccharide, were: 4.3 x 10(5) (MAST 83), 1.0 x 10(5) (MAST 107), 1.3 x 10(5) (MAST 107 Fab), 4.5 x 10(4) (MAST 108) and 1.9 x 10(5) l/mol (MAST 112). The results suggest that each epitope encompasses the O:4 specifying D-abequosyl residue together with different numbers of saccharides varying in size from di- to tetrasaccharides from the linear backbone chain. The antibodies also bind to different surfaces of the O-polysaccharide chain as suggested by its conformation.