Cross-reactivities of polyclonal antibodies against extensin precursors determined via elisa techniques

Abstract

We raised four sets of rabbit polyclonal antibodies against two highly glycosylated extensin precursors, P1 and P2, before and after hydrogen fluoride-deglycosylation. Use of an indirect non-competitive sandwich ELISA technique to determine antibody-antigen cross-reactivities revealed three epitope classes: 1. Glycosylated; 2. Nonglycosylated in the intact glycoprotein; 3. Exposed only after deglycosylation. Thus polyclonals raised against glycosylated P1 or P2 cross-reacted highly (50 %) with the heterologous glycosylated antigen, i.e. antibody-antigen pairs P1 /P2 and P2/P1, but much less with the deglycosylated antigens dP1 and dP2 (< 25 %), implying that the major epitopes are glycosylated; these probably correspond to hydroxyproline oligoarabinosides. The free sugars d-glucose, d-galactose and l-arabinose did not inhibit antibody-antigen binding, in contrast to free hydroxyproline arabinooligosaccharides which did compete at high levels (20–50 mM). Cross-reactivities towards other related macromolecules were low but positive for the following antibody/antigen pairs: P 1/potato lectin, P2/AGP, but negative towards larch arabinogalactan. Polyclonals dP1 and dP2 (raised against the deglycosylated precursors dPl and dP2 crossreacted significantly with their homologous glycosylated antigen (reactions dP1/P1 and dP2/P2), but only slightly with their heterologous antigen (reactions dP1/P2 and dP2/P1). These results imply that nonglycosylated epitopes of the glycosylated antigens P1 and P2 differ markedly from one another, and therefore corroborate primary structure information suggesting Val-Lys-Pro-Tyr-His-Pro as the major nonglycosylated epitope of P1 and Val-Tyr-Lys-Tyr-Lys as the major nonglycosylated epitope of P2.