In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR
Figure 3
Model experiments of in vitro selection of Fab fragments using mRNA display and emulsion PCR. (a) Confirmation of the H and L chain gene linking by PCR. The H chain or/and L chain gene of anti-fluorescein Fab fragments were amplified by PCR with forward and reverse primers of the corresponding gene or only reverse primers of both genes and analyzed on a 1.5% agarose gel. Primers H-F, H-R, L-F and L-R are UnivOL1-F, Myc-R, UnivOL2-F, and FLAG-R, respectively (Table 1). (b) Antigen-specific enrichment of mRNA-displayed Fab fragments. A single round of affinity selection was carried out for antigen-immobilized and nonimmobilized beads from a mixture of equally amounted anti-p53 and anti-fluorescein Fab fragment genes. The amount of each gene before and after selection was quantified by quantitative PCR. The amount of the positive control gene divided by that of the negative control gene is plotted for both before selection (gray) and after selection by antigen-immobilized bead (black) and nonimmobilized beads (white). (c) Confirmation of H and L chain gene linking by cloning and sequencing after affinity selection. mRNA-displayed anti-fluorescein Fab fragments and mRNA-displayed anti-p53 Fab fragments were mixed in a ratio of 1 : 50 and then used for affinity selection against fluorescein-immobilized beads. After selection, a total of 13 clones were sequenced, and the correspondence of the H and L chains was confirmed.
