Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa.
Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.
Martins
Sónia
S
Centro de Investigaçáo de Engenharia Química e Biotecnologia do Instituto Superior de Engenharia de Lisboa, Lisbon, Portugal.
Karmali
Amin
A
Andrade
Jorge
J
Custódio
Ana
A
Serralheiro
Maria Luísa
ML
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D013912:Threonine; D012965:Sodium Chloride; D000581:Amidohydrolases; C019665:amidase