SELECTION PROCEDURES FOR NONMATURED PHAGE ANTIBODIES - A QUANTITATIVECOMPARISON AND OPTIMIZATION STRATEGIES

Libraries of peptides and proteins can be displayed on the surface of filamentous bacteriophage. The efficient capturing of phage recognizin g a defined target molecule remains a serious obstacle, in particular when the phage are present at a low frequency or have a reduced affini ty like nonmatured phage antibodies and when the availability of targe t molecules is limited. We present theoretical considerations and expe rimental data which allowed us to substantially improve microselection under these conditions. We used a model phage displaying an anti-(2-p henyl-5-oxazolone) single-chain Fv antibody fragment. Following standa rd protocols and aiming at a low nonspecific binding, only 3.6 X 10(-3 )% of the phage input could be recovered from a single round of select ion performed in the wells of a microtiter plate. Our results explain why this often employed panning in wells is not efficient, especially with high-molecular-weight target molecules. We devised a procedure wh ich increased the probability of microselection by a factor of 34. An alternative capturing method using immunotubes with a new protocol dec reased the amount of required work by a factor of 30. In the case of a nonlimited supply of target molecules, column-affinity chromatography is recommended. (C) 1995 Academic Press, Inc.