Use of phage display and high-density screening for the isolation of an antibody against the 51-kDa subunit of complex I.
Monoclonal antibodies play an increasingly important role in structural biology. In this report, we develop the use of phage display technology for the isolation of an antibody that binds to a specific subunit of a macromolecular assembly. Antibodies that bind to the intact complex are selected from a phage display library and screened with a high-density Western blot to identify a subunit-specific binder. Conventional Western blotting and competition ELISA are then used to confirm the identity of the target subunit and that the antibody binds to the native protein complex and not to an epitope that is only revealed when the antibody is immobilized for phage selection. Using this technique, monoclonal scFv and Fab fragments have been produced that bind to the 51-kDa subunit of bovine complex I, a large integral membrane protein complex from mitochondria.
Rubinstein
John L
JL
MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK.
Holt
Lucy J
LJ
Walker
John E
JE
Tomlinson
Ian M
IM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000302:isolation & purification; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D042967:Electron Transport Complex I / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D015723:Gene Library; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D008929:Mitochondria, Heart / Q000201:enzymology; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D042967:Electron Transport Complex I