Monoclonal antibodies against native ant denatured forms of estrogen-induced breast cancer protein (BCEI/pS2) obtained by expression in Escherichia coli.
Several vectors were used to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS2) in Escherichia coli. The best results were obtained by using the pUR 290 expression vector after deletion of the sequence encoding the signal peptide of the protein. In these conditions, beta-galactosidase-BCEI/pS2 fusion protein accounted for approximately 20% of total proteins in bacterial extracts. It was purified by chromatography on DEAE-Trisacryl or by gel electrophoresis and electroelution. Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native protein and was very efficient for the immunoprecipitation and immunopurification of the protein from breast cancer cells; a second class recognized the denatured protein and was especially effective for immunoblot studies. BCEI/pS2 could be detected by immunocytochemistry in breast cancer biopsies using monoclonal antibodies on frozen or paraffin-embedded sections. One of the antibodies (mBCEI11) exhibited high affinity for the protein and could be used at 1.9 micrograms/ml concentration for immunolabeling of histological sections. The mBCEI11 antibody was used in immunoaffinity chromatography to purify the peptide in a single step from culture media of estrogen-treated MCF-7 cells.
Prud'homme
J F
JF
Groupe de Recherches sur la Biochemie Endocrinienne et la Reproduction (INSERM U. 135), Faculté de Médecine Paris-Sud, Le Kremlin-Bicétre, France.
Jolivet
A
A
Pichon
M F
MF
Savouret
J F
JF
Milgrom
E
E
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000473:pathology*; D002460:Cell Line; D002846:Chromatography, Affinity; D002872:Chromosome Deletion; D004273:DNA, Neoplasm / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C038865:estrogen regulated protein