Rapid screening for affinity-improved scFvs by means of single-molecule-PCR-linked in vitro expression

We have applied a combination of in vitro saturation mutagenesis and the single-molecule-PCR-linked in vitro expression (SIMPLEX) for rapid generation and screening of antibody mutants with improved affinity. A pool of anti-human serum albumin scFv mutant genes was created by in vitro saturation mutagenesis of a parent’s CDR-H3. Individual mutant genes were amplified by single-molecule PCR, and the PCR products were used directly as templates for in vitro coupled transcription/translation. Thereafter, all expressed scFv mutants were screened by competition ELISA. Two mutants showing improved affinities were selected and kinetically characterized. Because the single-molecule PCR, in vitro expression, and selection of distinct mutants were carried out parallelly in an array format, the process was very speedy and perfectly suited automation.