The development of monospecific antibodies against human thromboplastin apoprotein (apoprotein III) and their application in the immunocytochemical detection of the antigen in blood cells.

Human thromboplastin apoprotein (apoprotein III) purified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was purified a further 2-4 fold by PAGE in the presence of digitonin. Subsequent line immunoelectrophoresis of the protein revealed several lines, only one of which contained inhibitory antibodies. New inhibitory antibodies which were raised by using this particular line to immunize rabbits produced only a single line in immunoelectrophoresis of apoprotein III, with precipitated inhibitory antibodies being present only in the line. When these antibodies were used in electroblot immunobinding studies of crude thromboplastin after SDS-PAGE staining was found mainly in a single band of MW about 50,000, but also to some extent in immunologically related higher MW material. Prior deglycosylation of the thromboplastin using trinitrobenzenesulfonic acid resulted in a shift of the bulk of the main band representing an apparent MW reduction of 16%, and a corresponding shift in the position of protein with the capacity to bind inhibitory antibodies. Besides being a good criterion of specificity of the antibodies this also suggests that non-carbohydrate parts of apoprotein III may be involved in the interaction with Factor VII. Immunoperoxidase staining of unstimulated or endotoxin stimulated blood cells using the antibodies revealed the presence of significant amounts of apoprotein III only in stimulated monocytes, apparently available on the surface of the cells since it was detectable also by preembedding staining of fixed cells in suspension. The result is strong evidence that apoprotein III is synthesized de novo in monocytes upon endotoxin stimulation.