J.urmd '~t Immunoh~glt al Method~. 60 I 9831 351 - 358 35 I Elsexier A Convenient Enzyme-Linked Immunosorbent Assay for Testing whether Monoclonal Antibodies Recognize the Same Antigenic Site. Application to Hybridomas Specific for the fl_~-Subunit of Escherichia colt Tryptophan Synthase Bertrand Friguet ~, Lisa Djavadi-Ohaniance ~, Jacqueline Pages -~, Alain Bussard z and Michel Goldberg i I ~/tllt~ de Btoc htmte de~" Reguhltlom Cellalatres and -" L'mte d'lmmunologle Celhdalre. lnstltat Pasteur. 28 rue du Dot It'ur R,,u ~,. "fi'24 Parts CEDEX 15. Franc e ( Rece~.ed 27 .lull, 1982. accepted 4 January, 1983) Se~.en hxbr~doma clones, produong antibodies directed against the flz-,,ubumt of Es~her, hta toh tr3ptophan s3nthase, ha~e been obtained front mou,e cells. To te',t whether the corresponding mono- clonal antibodies recogmze different epltopes on f12. an ELISA double anttbod?, binding s~,tem has been de~eloped and i', reported here. The anttgen t~, first coated onto a microtitrat~on plate. Two monoclonal antibodies are then added etther ~eparatel,. or ~lmultaneou.,,13. and the amount of bound anttbod,, ts quantitatl,.el~, measured b 3 use of mmlunoglobuhn lrabb.t anti-mouse [gG) linked to .8-galacto,,ida,.e Addtt~xtt~ of the bound enz~,mat~c acti'~tt,, ~,, ob,,erxed '.'.hen the mc,noclonal anttbodtes brad to dtstmct epitope+. Using th~s test. it is +ho',,.n that. of the 7 anti-flz monoclonal antibodies, obtained. 5 recogmze the +ame ant~gemc .',tte and the 2 other~, recogmze a >econd +ire Ke} ~ords: monoclomd anttbodt -- double wltlbodt bm,hn.t,, -- enztme-/m/,ed tmmanosorhent assay -- spe¢tltt lit" lesl -- Irvfltophan st nthase Introduction For biological studies involving a highly ~pecific immunochemical reagent, one monoclonal antibody of good affinity is usually sufficient to establish the experimen- tal procedure. There are, however, circumstances x~here it is necessar,,, to use a series of monoclonal antibodies, all directed against the same antigenic molecule, but having different epitope specificities. One of the difficulties in obtaining such a series is the lack of a simple screening method for rapidly deciding whether 2 antibodies, produced b~ 2 independent clones, recognize the same or distinct regions of the macromolecular antigen. 01)22-1759 83 $03.00 ,C 1983 Elsevier Science Publishers BA'.