Preparation and characterization of a monoclonal antibody against the protein LIGHT.
LIGHT (which is homologous to lymphotoxins, shows inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes [Genome Database designation, TNFSF14]), a newly identified member of the TNF superfamily, is up-regulated upon activation of T-cells. LIGHT plays an important role in the T-cell-mediated tumor and graft-versus-host disease via LIGHT/HVEM/LT beta R signaling. To prepare specific monoclonal antibody (MAb) against murine LIGHT, a fragment containing the extracellular domain of LIGHT was inserted into prokaryotic expression vector pET-32a(+). The his-tagged fusion protein was expressed in BL21(DE3) in the form of inclusion bodies. The fusion protein was purified and refolded on-column using immobilized mental affinity chromatography. Rat MAb against murine LIGHT was obtained with hybridoma technique and specific ELISA screening. Western blotting and flow cytometry assays showed that MAb 4C11 had specific binding ability with LIGHT protein in eukaryotic cells. Lymphocyte proliferation assays indicated that this MAb could co-stimulate the proliferation of T-cells. Thus, this MAb may be the basis for detection of LIGHT protein in tissue or cell and be beneficial for the study of LIGHT/HVEM/LT beta R pathway.
Fan
Kexing
K
International Joint Cancer Institute, Second Military Medical University, Shanghai, PR China.
Zhou
Qian
Q
Wang
Hao
H
Guo
Yajun
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity; D049109:Cell Proliferation; D006825:Hybridomas / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D017510:Protein Folding; D051381:Rats; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; C505915:Tnfsf14 protein, mouse; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; D014409:Tumor Necrosis Factor-alpha