Screening of a human antibody phage display library against a peptide antigen using stimuli-responsive bioconjugates

A synthetic antibody phage library (ETH-2) was screened against the MUC-1 peptide. Two standard methods were used, namely screening on immunotubes coated with the peptide antigen and on streptavidin-coated paramagnetic particles together with a biotinylated MUC-1 peptide. The results were compared with those obtained using a novel approach based on a stimuli-responsive bioconjugate of avidin and poly-(N-isopropylacrylamide), also in combination with the biotinylated peptide. The poly-(N-isopropylacrylamide)-based bioconjugate is soluble below a temperature of 30 degrees C in the aqueous solutions pertinent to this study, but precipitates rapidly once this temperature is surpassed. The process is reversible, and the bioconjugate redissolves once the temperature is lowered again. The stimuli-responsive bioconjugate was used to isolate specific phage from the library in several rounds of panning, consisting of repeated thermoprecipitation/redissolution cycles in a procedure known as "affinity precipitation." As far as we know, this is the first time a compound as big as a viral particle has been specifically captured by a stimuli-responsive bioconjugate. Compared to the two other investigated screening techniques, affinity precipitation of the phage led to a greater variety of genetically different specific phage, while the process was easy to handle and required no dedicated equipment safe for a centrifuge and a thermostated water bath.