Towards proteome-wide production of monoclonal antibody by phage display.
Sequencing of the human genome reveals that there are approximately 30,000 genes that encode an even greater number of proteins which comprise the human proteome. Characterization of gene products at the genome-wide scale requires the development of high throughput methods to generate temporo-spatial information on each and every protein in the cell under normal and pathological conditions. Monoclonal antibodies are important reagents for these studies. We have developed a method to generate human monoclonal antibodies by selecting phage antibody libraries directly on antigen blotted onto poly(vinylidene fluoride) membranes. Cellular proteins are first separated by two dimensional (2D) gel electrophoresis, Western blotted onto poly(vinylidene fluoride) membranes, and used to select phage antibody libraries. Monoclonal antibodies can be generated against individual protein spots on a 2D gel. The antibodies are functional in Western blotting, ELISA, and immunohistochemistry. Automation of this process should allow high throughput production of monoclonal phage antibodies against cellular proteins as well as proteins that are uniquely expressed under pathological conditions.
Liu
Bin
B
Department of Anesthesia, University of California, San Francisco, Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, CA 94110, USA. liub@anesthesia.ucsf.edu
Huang
Lan
L
Sihlbom
Carina
C
Burlingame
Al
A
Marks
James D
JD
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D000941:Antigens / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D016477:Artifacts; D001331:Automation; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D011145:Polyvinyls / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D012680:Sensitivity and Specificity; D005465:Fluorocarbon Polymers
D000911:Antibodies, Monoclonal; D000941:Antigens; D010446:Peptide Fragments; D019151:Peptide Library; D011145:Polyvinyls; D020543:Proteome; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers