Preparation Of Anti-human Sydecan-1 Monoclonal Antibody And Analysis Of Its Biological Characteristics

Sydecan-l is a member of a family of integral membrane heparin sulfate proteoglycans. It consists of a core protein with five putative glycoaminoglycan(GAG) bearing three heparan sulfate chains(HS) and two chondroitin sulfate chains(CS) attachment sites which are located in the ectodomain. HS binds a variety of extracellular ligands such as matrix components, cell-cell adhesion molecules, heparin binding factors, degradative enzymes, protease inhibitors and some serum proteins through their covalently attached heparan sulfate chains. Sydecan-1 is known to participate in the regulation of the interaction between cells and their environment as cell co-receptor, and to take part in organ development, vessel formation and tissue regenration.Now, the research about sydecan-1 mainly focus on the relationship between the expression of sydecan-1 and the formation, the proliferation, the migration, the homing and prognosis of tumor cells. Some studies have showed that Sydecan-1 can be as an important prognostic indicator in some tumors. For example, sydecan-1 is a new independent prognostic parameter in multiple myeloma and hasprognostic significance in head and neck carcinoma. Therefore, the research about anti-sydecan-1 mAb must have great significant theoretic and clinical value.In this study, human myeloma cells 8228, which expressed sydecan-1 strongly, were used to immunize Balb/C mice. The immunized murine splenocytes were fused with SP2/0 murine plasmcytoma by using polyethylene glycol(PEG), then hybridomas were cultured in HAT selection medium. The positive colone secreting anti-human sydecan-1 continuously and steadily was obtained after repeatedly screening and cloning. Through this procedure, one hybridoma cell line 4B3 that could secrete specific anti-human sydecan-1 mAb was obtained and stablished.For analysis of its characteristics, Affinity chromatography that was used to purify mAb from ascites and Fast-strip method analysis that determine the subclass of 4B3 and competitive inhibition test by using the PE-conjugated positive control mAb and western blot were used. The results showed that the mAb concentration was 2.9mg/ml, the subclass of 4B3 was mouse IgGl and the mAb 4B3 recognized the identical or similar epitope with PE-conjugated positive control mAb. The biological function study of mAb 4B3 indicated that the anti-sydecan-1 mAb 4B3 could induce homotypic aggregation and proliferation arrest in XG-land XG-2 cells. These data showed that anti-sydecan-1 mAb 4B3 might be of significant clinical value and deserves further study.