Preparation of tissue-specific monoclonal antibodies using purified endothelial membrane proteins from biotinylated pulmonary vasculature of rhesus monkey.
In the present study, we perfused the rhesus lung vascular bed in situ with sulfo-NHS-LC-Biotin to biotinylate its luminal surface membrane proteins. After homogenization, dialysis, and affinity chromatography, biotinylated endothelial membrane proteins were successfully isolated and characterized as enriched endothelial membrane proteins with no contamination of intracellular proteins. When they were used as immunogens to develop monoclonal antibodies (MAbs), three MAbs--TX111, TX112, and TX113--were obtained. Among them, TX111 was demonstrated to specifically bind to rhesus lung tissue by Western blotting and enzymelinked immunosorbent assay (ELISA)--that is, positively stained capillary endothelium of rhesus lung. The molecular weight of the corresponding antigen for TX111 was approximately 70 kDa under reducing conditions. TX111 also reacted with human lung homogenate, but not with rat lung homogenate. These results suggest that (1) the biotinylation method is applicable for isolating endothelial proteins in situ from large animals; (2) anti-human protein MAbs are likely to be obtained using monkey proteins; and (3) TX111 is potentially useful for pulmonary vascular targeting.
Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210-093, P.R. China.
Wei
W
Zhang
Ying-Jiang
YJ
Zhou
Zhi-Gang
ZG
Guo
Feng
F
Bi
Jing
J
Zhang
Pat
P
Kumar
Xiang-Yang
XY
Tan
Jian-Ning
JN
Liu
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D019921:Biotinylation; D002846:Chromatography, Affinity; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008168:Lung / Q000098:blood supply*; D008253:Macaca mulatta; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D013388:Succinimides / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D008565:Membrane Proteins; D013388:Succinimides; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D001710:Biotin