Background
Methods
scFv-phage library, bacterial strains, and components
Expression and purification of exotoxin A domain I
scFv phage library screening
Rounds | I | II | III | IV | V | VI | VII |
---|---|---|---|---|---|---|---|
Protein (μg/ml) | 375 | 250 | 150 | 120 | 150 | 150 | 150 |
Blocking buffers | %2 skim milk | %3 BSA | %2 skim milk | %3 BSA | %2 skim milk | %3 BSA | %2 skim milk |
% Tween 20 | %0.1 | %0.1 | %0.1 | %0.1 | %0.1 | %0.1 | %0.1 |
Washing numbers | 3 | 20 | 10 | 20 | 20 | 20 | 25 |
Phage incubation time (hrs) | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Investigation of the specificity of the phage clones by polyclonal phage ELISA
Selection of scFvs clones to ExoA-DI
Screening of clones via soluble fragment ELISA
Primer | Sequence | |
---|---|---|
Phen | Reverse | 5′-CAG GAA ACA GCT ATG AC-3’ |
LMB3 | Forward | 5′-CTA TGC GGC CCC ATT CA-3’ |
Expression of soluble anti exotoxin A scFv in E. coli
Assessment of the reactivity of recombinant scFv with exotoxin A
Production of P. aeruginosa native exotoxin A
Evaluation of the reactivity of recombinant scFV antibody with native exotoxin A
Results
Enrichment and specificity of anti-exotoxin A phages during biopanning rounds
Round | Input (pfu) | Output (pfu) | Enrichment (output/input) |
---|---|---|---|
1 | 3.52*1012 | 1.40*106 | 0.39*10−6 |
2 | 1.40*1012 | 1.94*109 | 1.38*10−3 |
3 | 2.52*1012 | 1.83*108 | 0.72*10−4 |
4 | 1.62*1012 | 1.80*108 | 1.11*10−4 |
5 | 2.908*1013 | 7.27*108 | 2.5*10−5 |
Determination of the binding of scFv phage clones to ExoA-DI by monoclonal phage ELISA
Sequence analysis of the positive clones
C9 sequence | MKYLLPTAAAGLLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRRAPGKGLEWVSTISSSGSATSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTASSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQITQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYNASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNAGPTTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGAA | |
---|---|---|
VL/VH | VL | VH |
CDR1 | GFTFSSYA | QSISSY |
CDR2 | ISSSGSAT | NASYLQSGVPS |
CDR3 | AKTASSFDY | QQSNAGPTT |