Selection and characterization of antibodies from phage display libraries against internalizing membrane antigens.
Macromolecular delivery systems require high target cell specificity and efficient intracellular uptake. Monoclonal antibodies (mAbs) have been shown to successfully meet these needs and should, due to their biological nature and thus minimal toxicity and limited immunogenicity, be optimal delivery vehicles for various macromolecules (e.g., toxins, drugs, oligonucleotides). Such antibodies could be retrieved from phage display libraries by carefully designed selection and screening methods. In this chapter, we provide protocols for the isolation of phage-derived antibodies reactive to cell surface receptors, which upon binding will induce receptor-mediated internalization of the antibody/receptor complexes. In addition, a protocol describing the identification of target antigens by immunoprecipitation (ip) of cell lysates and preparation of gel plugs for subsequent MALDI-TOF analysis is included. Furthermore, we suggest several techniques that could be employed to confirm the specificity as well as the drug delivery potential of isolated clones.
Fransson
Johan
J
Centocor Discovery Research - San Diego, 3210 Merryfield Row, San Diego, CA 92121, USA.
Borrebaeck
Carl A K
CA
D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology*; D001435:Bacteriophages / Q000276:immunology*; D004705:Endocytosis; D005434:Flow Cytometry; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D000954:Antigens, Surface