Automated panning and screening procedure on microplates for antibody generation from phage display libraries.
Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human gamma-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 gamma-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and beta-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 beta-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency.
Turunen
Laura
L
VTT Technical Research Centre of Finland, Espoo, Finland. Laura.Turunen@vtt.fi
Takkinen
Kristiina
K
Söderlund
Hans
H
Pulli
Timo
T
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000941:Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001331:Automation; D002417:Cattle; D002999:Clone Cells; D016172:DNA Fingerprinting; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D004958:Estradiol / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008280:Magnetics; D008403:Mass Screening / Q000379:methods*; D008863:Microspheres; D019151:Peptide Library; D012371:Robotics; D012710:Serum Albumin, Bovine / Q000276:immunology / Q000378:metabolism; D013739:Testosterone / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012710:Serum Albumin, Bovine; D013739:Testosterone; D004958:Estradiol