[Preparation and identification of human soluble sPD-L1 and its antibodies].
This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.
Xu
Li-Hui
LH
Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Chi
Xiao-Yun
XY
Li
Feng-Yao
FY
Jia
Qian-Tao
QT
Zha
Qing-Bing
QB
He
Xian-Hui
XH
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D060890:B7-H1 Antigen; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D000906:Antibodies; D015703:Antigens, CD; D060890:B7-H1 Antigen; C423236:CD274 protein, human; D007106:Immune Sera; D011994:Recombinant Proteins