| Author/Editor | Wozniak, Gordana | |
| Title | Izdelava imunodiagnostičnih reagentov na osnovi rekombinantnega humanega tkivnega inhibitorja metaloproteinaz TIMP-1 | |
| Type | monografija | |
| Place | Ljubljana | |
| Publisher | Medicinska fakulteta | |
| Publication year | 2003 | |
| Volume | str. 189 | |
| Language | slo | |
| Abstract | Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are critical effectors of extracellular matrix turnover in normal and pathophysiological processes. The TIMPs form a family of at least four members, whose primary function is regulation of the metalloproteinase action. In cancerous disease, high-level biosynthesis of TIMP-1 is the result of a host response to tumor invasion and reflects a tendency to neutralise the activity of MMP while preserving the integrity of extracellular matrix. This phenomenon correlates with poor survival, again suggesting that there may be an excess of MMP activity over TIMP-I in cancers of poor prognosis. This proves the informative value of TIMP-1 concentration in the serum and renders it an interesting target for the isolation of antibodies. To quantitate TIMP-1 in biological samples, a strategy for isolation of monoclonal antibodies was applied that employs a phage-displayed single-chain scFv. In order to obtain sufficient amounts of TIMP-I to use as an antigen, high-level expression in Pichia pastoris was achieved under the control of the AOX-I promotor. Purified protein antigen was then used for panning to achieve enrichment of specific phage from naive scFv library. In four subsequent panning rounds, antibody fragments that display specificity to TIMP-I were selected and the binding of monoclonal phage antibodies was demonstrated by an enzyme-linked immunosorbent assay. For the construction of an ELISA assay based completely on recombinant proteins, single-chain Fv sequences were fused to N-terminus of modified Escherichia coli alkaline phosphatase to produce a detecting agent, or to N-terminus of the antibody CL domain to serve as a capture antibody. (Abstract truncated at 2000 characters). | |
| Descriptors | METALLOPROTEINASES RECOMBINANT PROTEINS TRANSFORMATION, GENETIC ANTIBODIES, MONOCLONAL ENZYME-LINKED IMMUNOSORBENT ASSAY PICHIA ESCHERICHIA COLI CLONING, MOLECULAR BASE SEQUENCE POLYMERASE CHAIN REACTION |