Preparation and characterization of a monoclonal antibody against human B lymphocyte stimulator.
B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family. It is required for B cell development. Deregulation of BLyS was involved in the pathogenesis of B cell-related autoimmune diseases and multiple myeloma. To prepare monoclonal antibodies (MAbs) against BLyS, cDNA encoding soluble BLyS (sBLyS) was first amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers, and then inserted into a prokaryotic expression vector pET-30a. Right recombinant plasmid was expressed in Escherichia coli strain BL21(DE3), purified by nickel affinity chromatography. Isolated sBLys was used as an antigen to immunize mice. Splenocytes of one immunized mouse were fused with NS- 1. Hybridomas secreting antibodies against sBLyS were identified by ELISA. One positive clone was selected to produce antibody by injecting the hybridoma into the peritoneal cavity of mice. After collecting ascites, the antibody was purified by protein A affinity chromatography. Western blot and immunoflourescence demonstrated that the antibody could bind recombinant sBLyS and genuine membrane-bound BLyS (mBLyS) on U937. This MAb can be used as a detecting reagent to analyze the serum level of BLyS in patients with autoimmune diseases and the expression profile of mBLyS on multiple myeloma cells.
Sun
Jian
J
College of Pharmaceutical Science and Technology, Tianjin University, Tianjin, PR China. jsun@public3@bta.net.cn
Li
Yan
Y
Sun
Yingxun
Y
Yu
Ming
M
Feng
Jiannan
J
Shen
Beifen
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D053264:B-Cell Activating Factor; D018076:DNA, Complementary / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology*; D020298:U937 Cells
D000911:Antibodies, Monoclonal; D053264:B-Cell Activating Factor; D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; C505719:TNFSF13B protein, human; C505720:Tnfsf13b protein, mouse; D014409:Tumor Necrosis Factor-alpha