Prediction Of Epitopes And Production Of Monoclonal Antibodies Against Gastric H,K-Atpase

Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a
theoretical and experimental strategy based on prediction of linear epitopes by
molecular modelling followed by production of anti-peptide antibodies. By
analysing the alpha subunit sequence, we predicted several epitopes corresponding
to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal
antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in
RaLISA, immunohistochemistry and Western blots demonstrating that they recognize
the native and the SDS-denatured ionic pump and that the epitopes are located at
the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M.
Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with
ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity.
Monoclonal antibody 95111, whose epitope is mapped between residues C529 and
E561, competes with mAb HK543 but not with the other two. We suggest that the
95111 epitope is overlapping or very close to the HK543-553 sequence. Induction
of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb
HK543 epitopes but not that of mAb 786, supporting the fact that the fragment
E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.