Monoclonal antibodies to human stefin B and determination of their epitopes.
Monoclonal antibodies (MAbs) to human stefin B were developed and three of them were characterised. Stefin B was cleaved into four peptides which were later subfragmented to smaller peptides. Only two peptides, of 24 and 30 amino acids, could bind to MAbs. In one instance, two peptides that were not consecutive in the sequence were recognised by the same antibody, proving that the epitope was discontinuous. Location of the epitopes was further narrowed by measuring the binding of MAbs to the complex of stefin B with papain. A sandwich ELISA (enzyme-linked immunosorbent assay), which measures the concentration of free inhibitor, was developed. It confirms that two out of three MAbs bind to different sites of stefin B. On the basis of the crystal structure of complex of stefin B with papain, the surface, accessible to a sphere with a radius of 5 A which simulates the accessibility of variable regions of antibody was determined. From the difference between accessibilities of free stefin B and stefin B in complex, the epitope location was determined more accurately.
Kopitar-Jerala
N
N
Department of Biochemistry, Jozef Stefan Institute, Ljubljana, Slovenia.
Curin-Serbec
V
V
Jerala
R
R
Krizaj
I
I
Gubensek
F
F
Turk
V
V
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010206:Papain; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; C524994:CSTB protein, human; C524992:Cstb protein, mouse; D015891:Cystatins; D000939:Epitopes; D011994:Recombinant Proteins; D055313:Cystatin B; D010206:Papain