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The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.
- Source :
-
Journal of immunology (Baltimore, Md. : 1950) [J Immunol] 1984 Feb; Vol. 132 (2), pp. 809-15. - Publication Year :
- 1984
-
Abstract
- The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.
- Subjects :
- Animals
Antibodies, Monoclonal biosynthesis
Antibodies, Monoclonal physiology
Antigen-Antibody Reactions
Binding Sites, Antibody
Binding, Competitive
Chemical Phenomena
Chemical Precipitation
Chemistry
Complement Factor B metabolism
Complement Pathway, Alternative
Hemolysis
Humans
Hybridomas immunology
Oligopeptides metabolism
Rabbits
Antibodies, Monoclonal immunology
Complement Activating Enzymes immunology
Complement Activating Enzymes metabolism
Complement Factor D immunology
Complement Factor D metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0022-1767
- Volume :
- 132
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of immunology (Baltimore, Md. : 1950)
- Publication Type :
- Academic Journal
- Accession number :
- 6558106