Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy.
Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (V H 3 Heparan sulfate (HS) 1 represents a heterogeneous class of molecules within the group of glycosaminoglycans. It has been implicated in many basic cellular phenomena, such as cell growth, migration, and differentiation (1-4). HS binds and modulates various proteins, including growth factors and cytokines, enzymes, protease inhibitors, and extracellular matrix proteins. Studies involving specific enzymatic or chemical cleavage and subsequent analysis of the resulting oligosaccharides indicate the existence of many HS species and the presence of domain structures within the HS molecule (5-15). There are clues that specific monosaccharide sequences within the molecule dictate the specific features of a given species, e.g. a pentasaccharide for the binding of HS/heparin to anti-thrombin III and a preferential sequence for the binding of HS to bFGF (4, 16 -19). The appreciation of the structural diversity of HS species and its role in pathological conditions is strongly hampered by the lack of appropriate methodologies. Sequence strategies are not at hand, and specific antibodies, obvious tools for studying diversity, are difficult to raise. HS, and glycosaminoglycans in general, are almost nonimmunogenic, and consequently, only a few specific antibodies have been described (20, 21). To circumvent this, we adopted antibody phage display technology because this system allows one to generate antibodies against "self" antigens. We report here on the generation and application of three specific antibodies against HS species using this technique. We compared these antibodies with two described mouse monoclonal antibodies, with regard to immunostaining on sections of rat kidney, immunoreactivity toward various HS preparations, and reactivity with bFGF sites on HS. EXPERIMENTAL PROCEDURES MaterialsA "single pot" human semisynthetic phage library (22) (now officially named synthetic scFv library 1) was generously provided by Dr G. Winter, Cambridge University, Cambridge, United Kingdom. This library contains 50 different V H genes with synthetic random complementarity-determining region 3 segments, which are 4 -12 amino acid residues in length. The heavy chains are combined with a single light chain gene (DPL 16). The library contains Ͼ 10 8 different clones.Two Escherichia coli strains were used: the suppressor strain TG1 (K12, D(lac-pro), supE, thi, hsdD5/FЈtraD36, proA ϩ B ϩ , laqI q , lac-ZDM15), and the nonsuppressor strain HB2151 (K12, ara, D(lac-p...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.