Production and characterization of a monoclonal antibody to partially purified estrogen receptor from human breast tumor.
A hybridoma cell line that secretes monoclonal antibody, MAb-ER-Br-1-15-4-18 is established. The MAb is highly specific for estrogen receptor (ER) from human breast tumor cells. In order to raise the antibody, the ER was first isolated from human breast tumor. Mice were immunized with the partially purified ER and the fusion of the spleen cells from the mouse, showing the highest serum titer, with the cells of the NS-1 mouse myeloma line, produced hybrid cells which continuously secreted antibodies specific for ER. Three of the hybridoma cultures which tested strongly positive were cloned using limiting dilution method and one of the cell lines was selected for further study. The recovery of the MAb from the cell culture was done by ammonium sulfate precipitation followed by dialysis and then hydroxylapatite liquid chromatography using linear gradients. The purity of the antibody was checked by polyacrylamide gel electrophoresis. The MAb was isotyped and found to be IgG1. When checked against other antigens the MAb showed a minimal cross-reactivity to ER from rabbit uterus and none to ovalbumin or rat liver ferritin. Further experiments showed that the MAb recognized the ER bound to the hormone and ER in the nucleus of breast tumor cells.
Sardesai
S
S
Department of Oncology, Providence Hospital, Southfield, MI 48075.
Vaughn
C B
CB
Chen
Y M
YM
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D001943:Breast Neoplasms / Q000276:immunology*; D002467:Cell Nucleus / Q000032:analysis; D003600:Cytosol / Q000032:analysis; D006801:Humans; D007118:Immunoassay; D007132:Immunoglobulin Isotypes / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007132:Immunoglobulin Isotypes; D011960:Receptors, Estrogen