Isolating and engineering human antibodies using yeast surface display.
This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.
Chao
Ginger
G
Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue E19-563, Cambridge, Massachusetts 02139, USA.
Lau
Wai L
WL
Hackel
Benjamin J
BJ
Sazinsky
Stephen L
SL
Lippow
Shaun M
SM
Wittrup
K Dane
KD
D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008280:Magnetics; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling
D000906:Antibodies; D007135:Immunoglobulin Variable Region