Screening of clones by soluble fragment ELISA

The binding specificity of positive phage clones against EGFRvIII was examined by scFv ELISA. The positive clones confirmed by PCR were grown up in E. coli TG1 suppressor strain for producing soluble scFv in which all selected clones can produce scFv even clones containing TAG stop codon.

For this, the positive clones were grown up in 2xTY/ ampicillin/ 0.1 % glucose media at 37°C until the OD600 was approximately 0.9. At this stage, scFv expression was induced with 1mM IPTG and shaking was continued at 200 rpm overnight at 30°C. Soluble scFvs obtained from the periplasmic/osmotic fractions were then retested by the EGFRvIII -captured ELISA. Accordingly, EGFRvIII peptide with concentration of 25µg/ml was coated into the plate overnight at 4°C. After blocking with 3% BSA for 2hrs and washing with PBST, soluble scFvs were added to the plate in different dilutions and incubated for 1hr. After washing with PBS-T, the plate was incubated with HRP conjugated Protein L (1:2000 dilution in 1% BSA–PBS) for 1hr, and then washed. The reaction was developed with TMB substrate and the OD was read at 450nm.