A sensitive and rapid in situ immunoassay to quantitatively determine the cellular antigens in intact cultured cells.
A sensitive and rapid in situ immunoassay to quantitatively determine the cellular antigens in intact cells was developed. Antigens located in plasma membrane, endosome, cytosol, lysosome or endoplasmic reticulum of cultured cells were fixed in situ and reacted with monoclonal antibodies after permeabilization of cells with saponin. The antigen-antibody complexes were quantified by colorimetric method of peroxidase-substrate reaction. Epidermal growth factor receptor, monomer of pyruvate kinase M2 and the endoplasmic reticulum-associated thyroid hormone-binding protein were easily detected from 0.5-1 x 10(4) cells by this method. Antibody as low as 10 ng/ml gave reproducible results. Using this method, the in vivo dynamic interconversion of monomer-tetramer of pyruvate kinase M2 was found to be regulated by glucose. The ligand-induced epidermal growth factor receptor through different subcellular compartments during endocytosis was easily quantified by this method. This method was also used to compare the different amounts of the endoplasmic reticulum-associated thyroid hormone-binding protein in various cultured cells. Thus, the in situ immunoassay is an easy and versatile method which can be used to study various cellular antigens and their involvement in cellular processes.
Ashizawa
K
K
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Cheng
S Y
SY
D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002477:Cells / Q000737:chemistry*; D003600:Cytosol / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005916:Globulins / Q000302:isolation & purification; D006041:Goats; D006801:Humans; D007118:Immunoassay; D007150:Immunohistochemistry; D008247:Lysosomes / Q000737:chemistry; D015388:Organelles / Q000737:chemistry; D011233:Precipitin Tests; D012680:Sensitivity and Specificity; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000941:Antigens; D005916:Globulins