Colony lift assay using cell-coated filters: a fast and efficient method to screen phage libraries for cell-binding clones.
For efficient screening of phage antibody libraries obtained by selection on whole cells, we have developed a modified colony lift assay using cell-coated filters. Both cells growing in suspension as well as adherent cells can be coated onto nitrocellulose filters and used to detect bacterial colonies responsible for the production of cell-binding (specific) single chain variable fragment (scFv) antibodies. We demonstrate, using a selected library developed in our laboratory (named "AB" library) as a model system, that the frequency of specific clones as detected by colony lift assay using cell-coated filter is comparable to the frequency of positive clones as detected by the "classical" method (i.e. random picking and flow cytometric analysis). However, the colony lift assay enables detection and isolation of a higher number of specific clones as compared to the random pick. This is due to screening of a much higher number of clones simultaneously (it is possible to screen at least 1000 clones plated on one 9-cm agar dish). Using this method, clones occurring at a low frequency (such as present in early selection rounds) can be detected and isolated efficiently. We clearly demonstrate the usefulness of the colony lift assay with cell-coated filter by applying it to screen the head-and-neck carcinoma (HN) library (a selected library generated in our laboratory). Using the assay, but not the random picking, we were able to isolate specific clones from 2nd to 3rd selection rounds of the HN library.
Radosević
Katarina
K
Department of Immunology, Erasmus MC, University Medical Center Rotterdam, The Netherlands. k.radosevic@crucell.com
Voerman
Jane S A
JS
Hemmes
Annabrita
A
Muskens
Femke
F
Speleman
Luciënne
L
de Weers
Michel
M
Rosmalen
Judith G M
JG
Knegt
Paul
P
van Ewijk
Willem
W
D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D018914:Cells, Immobilized; D002999:Clone Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D007158:Immunologic Techniques; D051379:Mice; D019151:Peptide Library
D007128:Immunoglobulin Fragments; D019151:Peptide Library