Epitope mappingofanti-proteinase 3andanti-myeloperoxidase antibodies

SUMMARY Anti-proteinase 3(PR3)andanti-myeloperoxidase (MPO)autoantibodies are presentinmany patients withWegener's granulomatosis (WG)andmicroscopic polyarteritis. Theaimofthis study was todetermine whether these antibodies boundtolinear peptide sequences on their target antigens. Ifcommon linear epitopes were demonstrated, thenthese couldbemanufactured and usedindiagnostic ELISAsforanti-PR3 andanti-MPO antibodies. Inaddition, any homology between these epitopes andbacterial orviral sequencesmightimplicate those microorganisms in thedevelopment ofthese antibodies andthepathogenesis oftheassociated diseases. Thepresence oflinear epitopes on PR3 andMPO was suggested bythebinding ofthecorresponding autoantibodies tothese proteins after theyhadbeenreduced withbeta-mercaptoethanol (3-ME) anddenatured withSDSorboiling, anddigested with proteases. Fourofthe22serawithanti-PR3 antibodies boundtoPR3inWestern blots after treatment withSDS,,(-ME andboiling for 5min. Thermal denaturation reduced theamountofbinding more thanother forms ofdenaturation. One serum withanti-PR3 antibodies boundtoLys-CandGlu-C-digested PR3indotblots. Linear epitopes couldnotbefurther defined bytheir binding inan ELISAusing overlapping peptides corresponding tothePR3molecule because ofnon-specific binding. Threeofthefive serawith anti-MPO antibodies boundtoMPO inWestern blots after treatment withSDS,,3-ME andboiling for5min.Oneserum withanti-MPO antibodies boundtoLys-CandGlu-C-digested MPO indot blots. Again, linear epitopes couldnotbefurther defined using an ELISAwithoverlapping peptides because ofnon-specific binding. Someanti-PR3 andanti-MPO antibodies arelikely to recognize linear epitopes, butthese cannotbedefined byuseofa PINELISAsystem.