Monoclonal antibodies recognize conformational epitopes on wild-type and recombinant mutant amidases from pseudomonas aeruginosa.
Hybridoma technology was used to raise monoclonal antibodies (MAbs) against wild-type amidase from Pseudomonas aeruginosa. 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 T103I 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 T103I amidase than for the wild-type enzyme. However, this MAb did not recognize either wild-type or mutant T103I 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 C91A 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 (T103I) activity. However, this MAb did not affect mutant C91A 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 (T103I and C91A) amidases.
Martins
Sónia
S
Centro de Investigação de Engenharia Química e Biotecnologia, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, Lisbon, 1950-062, Portugal.
Lourenço
Sara
S
Karmali
Amin
A
Serralheiro
Maria Luísa
ML
D000581:Amidohydrolases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D002474:Cell-Free System; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D009154:Mutation / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019665:amidase