Preparation of monoclonal antibodies against different epitopes on extracellular domain of human receptor for advanced glycation end product

Abstract

Abstract: Objective To prepare and characterize monoclonal antibodies (mAb) against recombinant human receptor for advanced glycation end product (rhRAGE). Methods BALB/c mice were immunized with recombinant extracellular domain(amino acid 23-342) of human advanced glycation end product (RAGE), and hybridoma was generated with mouse spleen cells and myeloma NS-1 cells. After three fusions and cloning, two hybridoma cell lines secreting monoclonal antibodies to RAGE were obtained and monoclonal antibodies were purified from the ascites followed by characterization with indirect enzyme-linked immunosorbent assay (ELISA), flow cytometry and Western blotting. Results and Conclusion Two hybridoma cell lines secreting anti-RAGE mAb were established and designated as B2.2 and E10, respectively. Both of mAb B2.2 and E10 belonged to IgG2b isotype and could bind to recombinant RAGE and natural RAGE expressed on THP-1 cells. In addition, B2.2 and E10 could recognize different epitopes of RAGE, which were conformed to be capable of detecting soluble recombinant RAGE in sandwich ELISA. These two mAbs against different epitopes of rhRAGE would be useful for study of glycation end product and RAGE-related diseases.

CLC Number:

R392.11

ZHU Ping¹, TANG Lei¹, ZHAO Shan-chao², LU Xiao¹, HOU Fan-fan², FU Ning¹. Preparation of monoclonal antibodies against different epitopes on extracellular domain of human receptor for advanced glycation end product[J]. Journal of Southern Medical University, 2004, 24(02): 129-132.

References

[1] Brett J, Schmidt AM, Yan SD, et al. Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues[J]. Am J Pathol, 1993, 143(6): 1699-712.
[2] Hori O, Yan SD, Ogawa S, et al. The receptor for advanced glycation end-products has a central role in mediating the effects of advanced glycation end-products on the development of vascular disease in diabetes mellitus[J]. Nephrol Dial Transplant, 1996,11 (Suppl 5):13-6.
[3] Miyata T, Hori O, Zhang J, et al Thereceptor for advaneed glycation end products (RAGE) is a central mediator of the interaction of AGE-beta2 microglobulin with human mononucleεr phagocytes via an oxidant-sensitive pathway. Implications for the pathogenesis of dialysis-related amyloidosis[J]. J Clin Invest, 1996, 98(5): 1088-94.
[4] Sasaki N, Toki S, Chowei H, et al. Immunohistochemical distribution of the receptor for advanced glycation end products in neurons and astrocytes in Alzheimer’sdisease[J]. Brain Res, 2001, 888 (2):256-62.
[5] Chappey O, Dosquet C, Wautier MP, et al. Advanced glycation end products, oxidant stress and vascular lesions[J]. Eur J Clin Invest,1997, 27(2): 97-108.
[6] Hsieh HL, Schafer BW, Sasaki N, et al. Expression analysis of S100 proteins and RAGE in human tumors using tissue microarrays[J].Biochem Biophys Res Commun, 2003, 307(2): 375-81.
[7] Ishihara K, Tsutsumi K, Kawane S, et al. The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site[J]. FEBS Lett, 2003, 550(1-3): 107-13.
[8] Hou FF, Chertow GM, Kay J, et al. Interaction between β 2m and AGE in the development of dialysis-related amyloidnsis[J]. Kidney Int, 1997, 51(5): 1514-9.
[9] Wautier JL, Zoukourian C, Chappey O, et al. Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy, soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rat[J]. J Clin Invest, 1996, 97(1): 238-43.