Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.
We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.
Mersmann
M
M
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Schmidt
A
A
Tesar
M
M
Schöneberg
A
A
Welschof
M
M
Kipriyanov
S
S
Terness
P
P
Little
M
M
Pfizenmaier
K
K
Moosmayer
D
D
D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001435:Bacteriophages; D016367:Baculoviridae / Q000235:genetics; D036022:Capsid Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology; D013382:Subtraction Technique; D013745:Tetanus Toxoid / Q000276:immunology; D014760:Viral Fusion Proteins / Q000235:genetics*
D000907:Antibodies, Bacterial; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D013745:Tetanus Toxoid; D014760:Viral Fusion Proteins; C055966:immunoglobulin Fv