[Comparison of site-mutated and error-prone PCR methods for constructing the secondary phage antibody libraries]

To compare site-mutated PCR and error-prone PCR methods in constructing the secondary phage antibody libraries derived from a primary extracellular domain of cell adhesion molecule L1 (L1-ecd) binding single-chain variable fragment (scFv) antibody.

Secondary mutant phage libraries were established by transfecting the mutated phage at the DNA level to E.coli TG1 with designed site-mutated PCR or error-prone PCR primers. Using the selected phagemid as the template, the mutated plasmid was amplified by PCR and then constructed with restriction enzyme cutting and ligation. Phage-based ELISA was used to calculate the ratios of the positive monoclones from the two libraries and the results were statistically compared using the Pearson x(2); method.

The size of the two libraries were 1.4×10(6); pfu/mL (site-mutated library) and 2.5×10(6); pfu/mL (error-prone library), respectively. The ratios of positive clones were 32.5% and 35.5%, respectively. The P value was 0.67, showing no significant difference.

These two methods can be widely used to obtain antibodies with a high affinity on the basis of the existing phage antibody.