A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein.
A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.
Reed
J C
JC
Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia 19104-6082.
Tanaka
S
S
Cuddy
M
M
Cho
D
D
Smith
J
J
Kallen
R
R
Saragovi
H U
HU
Torigoe
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology*; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases