New method for determination of average scFv fragment number displayed on the M13 phage surface

Abstract

Single-chain-Fv (scFv) display M13 phage library has been regarded as a powerful tool for screening specific antibodies via binding with target proteins. Generally, the library quality is evaluated through detecting gene fragments by molecular biology methods, which is not only time- and labor-consuming, but also impossible to obtain quantitative information about the binding capacity of the phage library. In our recent study, a new method to calculate the average scFv number displayed on the M13 phage surface was proposed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. By this method, enhanced green fluorescent protein (EGFP) and scFv phage clones that could specifically bind with EGFP were mixed with different ratios, followed by analysis by CE-LIF. With the dilution of EGFP by phage solution, the peak areas of scFv phage clones and free EGFP were decreased continuously, while that of the EGFP-M13 phage complex was found to decrease initially, then trend to be stable, and finally decrease further. When the volume ratio of the M13 phage to EGFP reached 660:1, corresponding to the molecule number ratio as 1:2.6, no more EGFP was found to bind with the M13 phage, which demonstrated that, by average, 2.6 scFv fragments that could bind with EGFP were displayed on the M13 phage surface. All these experimental results demonstrated that, by such a method, the quantitative evaluation of the phage library could be achieved with high throughput and accuracy.