Monoclonal antibodies recognize conformational epitopes on wild-type and recombinant mutant amidases from XXXX .

@entity36575 @entity70756 @entity796
Hybridoma technology was used to raise monoclonal antibodies (MAbs) against wild-type amidase from @entity796 . Hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly. but release under mild- and non-denaturing elution conditions, which can be used as ligands in immunoaffinity chromatography. Two of these hybridoma clones (C9E4 and B1E4) secreting MAbs against wild-type amidase were selected in order to check if they are PR-MAbs by using ELISA-elution assay. These hybridoma cell lines secreted MAbs of IgG class which were purified in a single step by Protein A-Sepharose CL-4B chromatography, which revealed two protein bands on SDS-PAGE. Specificity studies of MAb C9E4 revealed that it recognized a common epitope on wild-type and mutant @entity36575 amidases as determined by direct ELISA, as well as by Western blotting under native conditions. This MAb exhibited a higher-affinity constant (K) for the mutant @entity36575 amidase than for the wild-type enzyme. However, this MAb did not recognize either wild-type or mutant @entity36575 enzymes under denaturing conditions suggesting that it binds to a conformation-sensitive epitope on amidase molecule. On the other hand, it also does not recognize either native or denatured forms of mutant @entity70756 amidase suggesting that this substitution disrupted the conformational epitope present on amidase molecule. Furthermore, MAb C9E4 inhibited about 80% of wild-type amidase activity, whereas it activated about 80% of mutant amidase ( @entity36575 ) activity. However, this MAb did not affect mutant @entity70756 amidase activity which is in agreement with other results presented in this work. The data presented in this work suggest that this MAb acts as a powerful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant ( @entity36575 and @entity70756 ) amidases.
