Two-site immunoenzymometric assay for the 52-kDa cathepsin D in cytosols of breast cancer tissues.
We developed a solid-phase two-site immunoenzymometric assay (IEMA) of the estrogen-induced 52-kDa cathepsin D (EC 3.4.23.5) and its processed forms (48-kDa and 34-kDa proteins) in cytosols of breast cancer tissues, using two monoclonal antibodies directed against two different epitopes of these antigens. The first antibody is bound to a polystyrene microtiter well; the second is labeled with alkaline phosphatase. The assay involves a simultaneous incubation of the antigen with both antibodies, because we observed signal loss during sequential incubations. Alkaline phosphatase was chosen because other enzymes (peroxidase, beta-galactosidase) were inhibited by cytosol extraction buffers. The measurable range of 52-kDa-related proteins is from 0.3 to 6 nmol/L with precision (CVs) within and between runs of 3.9% and 15.8%, respectively. The sensitivity, accuracy, and rapid turnaround time of the two-site IEMA should facilitate the clinical evaluation of this new marker in oncology.
Rogier
H
H
Sanofi Recherche, Montpellier, France.
Freiss
G
G
Besse
M G
MG
Cavalié-Barthez
G
G
Garcia
M
M
Pau
B
B
Rochefort
H
H
Paolucci
F
F
D000469:Alkaline Phosphatase; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000276:immunology; D001943:Breast Neoplasms / Q000032:analysis*; D002402:Cathepsin D / Q000032:analysis*; D003600:Cytosol / Q000032:analysis; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D008970:Molecular Weight; D011786:Quality Control
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes; D000469:Alkaline Phosphatase; D002402:Cathepsin D