Abstract
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 (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene, DP-42), and YYHYKVN (VH1 gene, DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 microM. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- and N-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.
Highlights
Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies
The VH family and germ line segments are VH3 and DP-38 for HS4C3, VH3 and DP-42 for HS4D10, and VH1 and DP-8 for HS3G8
We describe the generation of three anti-HS antibodies from a semisynthetic phage display library
Summary
A “single pot” human semisynthetic phage library [22] ( officially named synthetic scFv library 1) was generously provided by Dr G. An additional negative selection step was introduced In this case, after the second round of selection, 1 ml of phages was added to a tube containing 3 ml of PBS containing 30 g of dermatan sulfate and 30 g of chondroitin 4-sulfate and incubated for 1 h at 22 °C under continuous rotation. After the second round of selection, 1 ml of phages was added to a tube containing 3 ml of PBS containing 30 g of dermatan sulfate and 30 g of chondroitin 4-sulfate and incubated for 1 h at 22 °C under continuous rotation Two ml of this suspension was subjected to two additional rounds of panning on HS.
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