Monoclonal antibodies reacting with placental protein 5: use in radioimmunoassay, Western blot analysis, and immunohistochemistry.
Monoclonal antibodies were raised reacting with placental protein 5 (PP5), a glycoprotein with properties of a serine protease inhibitor. Immunization was carried out with an antigen purified from late pregnancy placenta tissues. After fusion with myeloma cells, clones producing antibodies reacting with PP5 were isolated. Antibodies produced by two of the established hybridoma clones were characterized. The Ka of the antibodies was 0.22 x 10(9) L/mol and 0.3 x 10(8) L/mol. in Western blot analysis, both monoclonal antibodies reacted with the purified antigen that had a relative molecular weight (Mr) of 30 kd, but minor components of Mr 27 kd, 56 kd, and 62 kd were also identified. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions, the purified protein yielded three polypeptides (Mrs of 16.4 kd, 16.8 kd, and 18.3 kd) that did not react with the monoclonal antibodies in Western blot analysis. By immunoperoxidase staining with monoclonal and polyclonal antibodies, PP5 was localized to the syncytiotrophoblast, cytotrophoblast, and endothelium of early and late pregnancy placenta tissues, whereas various other tissues were PP5-negative. In immunofluorescence staining, isolated endothelial cells were stained with both monoclonal antibodies. Endothelial cells in monolayer culture released into the medium a substance that is immunologically similar to purified PP5.
Bützow
R
R
Department I of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland.
Virtanen
I
I
Seppälä
M
M
Närvänen
O
O
Stenman
U H
UH
Ristimäki
A
A
Bohn
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D010920:Placenta / Q000276:immunology*; D011257:Pregnancy Proteins / Q000276:immunology / Q000378:metabolism*; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D011257:Pregnancy Proteins; C021240:tissue-factor-pathway inhibitor 2