Determination of human factor VIII (AHG-associated protein)--antigen in endothelial cells from Xenopus laevis (XTH cells). Evaluation of a sensitive ELISA technique.
AHG-associated protein (AHG-a.p.), the antigen of the blood-clotting factor VIII complex, is a specific endothelial cell marker. Primary (p-XTH) and established (XTH-2) endothelial cells from the hearts of Xenopus laevis tadpoles were assayed for the presence of this marker by means of immunological cross-reaction (recognition of common antigenic sites) with antiserum against human AHG-a.p. Radial immunodiffusion and rocket immunoelectrophoresis proved to be insufficiently sensitive, whereas immunofluorescence and a newly evaluated ELISA technique gave positive results. The very high sensitivity of the ELISA (less than 1/240,000 of the AHG-a.p. in 0.1 ml human standard plasma can be detected) and the removal of interfering proteins by gel filtration also revealed the presence of AHG-a.p. in the fetal calf serum used in the culture medium; earlier investigations into this subject by a one-step radioimmunoassay had reported negative results. Specially adapted XTH-2 cells were grown in a protein-and serum-free hydrolysate medium in order to demonstrate the presence of a Xenopus-derived antigen that was immunoreactive with the anti-human AHG-a.p.
D000818:Animals; D004727:Endothelium / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D009206:Myocardium / Q000166:cytology; D014981:Xenopus
D005169:Factor VIII