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 l inear epitopes by molecular modelling followed by production of anti-p eptide antibodies. By analysing the a subunit sequence, we predicted s everal epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786, A ll three react against gastric H,K-ATPase in RaLISA, immunohistochemis try 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 a greement with ATP binding to K519, Neither mAb 543, nor mAb 786 inhibi t the ATPase activity, Monoclonal antibody 95111, whose epitope is map ped between residues C529 and E561, competes with mAb HK543 but not wi th the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by bin ding FITC to K519 increases the number of mAb 95111 and mAb HK543 epit opes but not that of mAb 786, supporting the fact that the fragment E5 43-Y553 changes accessibility, maybe during the E1-E2 transconformatio n.