Figure 3

Screening of specific scFvs using a microarray format. Cell free-expressed proteins were labeled with 647 AlexaFluor and incubated with GFP- and Trx-specific scFvs microarrays to identify highly specific scFv binders. E. coli-expressed proteins were used as a control. A) Selection of GFP-specific scFvs. Left, performance of the microarray with cell free-expressed 647-labeled GFP or Trx as control. Right, performance of the microarray with E. coli expressed proteins followed by a polyclonal anti-GFP or a monoclonal anti-Flag and by AlexaFluor 555 labeled antibodies gave a green fluorescent signal. White boxes: 10 scFv antibodies against GFP showing at least 3-fold higher fluorescence signal than the control Trx values. Red boxes: scFvs showing non-specific binding for GFP. B) Anti-GFP scFv clones were tested by ELISA using GFP and GST to compare the microarray technology with ELISA screening. C) Selection of Trx-specific scFvs. Left, performance of the microarray with cell free-expressed 647-labeled Trx or GFP as control. Right, performance of the microarray with E. coli-expressed proteins followed by a polyclonal anti-GFP antibody or a monoclonal anti-Flag and by AlexaFluor 555 labeled antibodies. White boxes: 8 reactive scFv antibodies detected by antibody microarrays against Trx showing at least 3-fold higher fluorescence signal than the control GFP values. Red boxes: scFvs showing non-specific binding for Trx. D) Anti-Trx scFv clones were tested by ELISA using Trx, GST-His6 tagged and BSA to compare the performance of the microarray technology to identify Trx scFv binders with ELISA screening. Green arrows indicate the top three scFvs that gave the highest microarray signal for GFP or Trx and were further validated by other immunological techniques.