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Figure 5: Effects of Ex-phage packaging on antigen-binding reactivity of scFv displayed on recombinant phage. (A) Determination of scFv:pIII fusion protein expression by immunoblot. Recombinant phage particles were obtained by infecting JS5 cells carrying pIGT3-hsp70 phagemid with either M13KO7 helper phage (pIGT3/ M13KO7) (lane 1) or Ex-phage (pIGT3/ Ex-phage) (lane 2). Phage proteins were separated by loading approximately 1010 recombinant phage into each lane of 10% SDS–PAGE, and immunoblot was carried out with anti-gIII mAb to determine the amount of scFv:pIII fusion proteins displayed on the surfaces of phage particles. Goat anti-mouse IgG antibody conjugated with HRPO was used for the secondary antibody. (B) Antigen-binding specificity of recombinant phage packaged with either M13KO7 or Ex-phage. BSA, lysozyme (Sigma Co.), recombinant GST or recombinant human HSP-70 were coated in microtiters, and 1010 scFv phage packaged with either M13KO7 or Ex-phage were applied to each well for phage ELISA. The same amount of M13KO7 helper phage was used as a negative control. The bound phage were detected with anti-M13 antibody conjugated with HRPO. The binding signal was analyzed at OD405. (C) Antigen-binding sensitivity of phage particles packaged with either M13KO7 or Ex-phage. Phage ELISA was performed as described above except that serial dilutions of purified human HSP-70 fusion protein were used to coat microtiter plates.

Image Text (High Precision): 0.1 405 A.D. Ex TG1 antigen concentrations fusion lysozyme m13k07 pIGT3 pIGT3/Ex pIII phage preparations scFv

Other Images from "An improved helper phage system for efficient isolation of specific antibody molecules in phage display":


Figure 4 Diagram of Ex-phage system. Ex-phage are...

Figure 6 Enhancement of panning efficiency by Ex-...

Figure 3 Detailed diagram of pIGT3. Two different...

Figure 5 Effects of Ex-phage packaging on antigen...

Figure 1 Mutagenesis of M13KO7 helper phage genom...

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Abstract

Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F+ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.


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