Monoclonal antibodies against Pseudomonas aeruginosa elastase: a neutralizing antibody which recognizes a conformational epitope related to an active site of elastase.
We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.
Yokota
S
S
Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co. Ltd, Hyogo, Japan.
Ohtsuka
H
H
Noguchi
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015153:Blotting, Western; D002614:Chelating Agents; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D000939:Epitopes; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases