Monoclonal antibodies as probes to detect conformational changes in the rat cysteine proteinase inhibitor cystatin A.
Five monoclonal antibodies (MAbs), 77, 114, 138, 175 and 187, were established for rat cystatin A. MAbs 77, 114, 138 and 175 were shown to belong to the IgG1 subclass, whereas MAb 187 was an IgM. These MAbs partially suppressed inhibitory activity of rat cystatin A to papain. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs with NH2-terminally truncated forms and fragments of rat cystatin A by an enzyme-linked immunosorbent assay (ELISA), and by reactivity with the inhibitor on immunoblotting. In competitive binding assays the MAbs did not compete with each other, indicating that the epitopes recognized by these MAbs were substantially different. The conformational epitope recognized by the three MAbs 114, 138 and 175 belonged to one group that was highly sensitive to denaturation, but those epitopes were unchanged by NH2-terminal truncation. MAb 187 was able to recognize a linear epitope present in amino acid residues 15-50 in the NH2-terminal region. MAbs 77 and 114 reacted weakly with mouse cystatin A but not at all with human cystatin A, whereas MAb 187 reacted similarly with mouse cystatin A but at about half that level with human. The MAbs produced in this study should be useful tools for detecting conformational changes in the rat cystatin A molecule.
Takeda
A
A
Department of Clinical Pathology, Fujigaoka Hospital, Showa University School of Medicine, Kanagawa, Japan.
Iwasawa
A
A
Nakamura
Y
Y
Omata
K
K
Nakaya
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001667:Binding, Competitive; D002460:Cell Line; D015891:Cystatins / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D000939:Epitopes; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010206:Papain / Q000187:drug effects; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D015891:Cystatins; D000939:Epitopes; D010446:Peptide Fragments; D010206:Papain