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If I'm using a bacteriophage for phage display and I'm trying to avoid avidity effects by using a helper phage what would be the best way to maintain a large library size while keeping everything monomeric? I'm asking in terms of maintaining an appropriate infection ratio and or phage tricks and tools to maintain the that stoichiometry.

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If you don't end up getting an answer here, I would recommend reposting on researchgate –  yingw Jan 18 '13 at 19:00
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1 Answer 1

I found article in Nature:

A helper phage to improve single-chain antibody presentation in phage display

Experimental protocol

Standard cloning procedures, determination of colony-forming units and plaque-forming units, and immunoblot after PAGE were carried out according to Sambrook et al.

Construction of the packaging cell line.

DH5alpha/pIII [M13KO7DeltapIII]. The M13 pIII gene fused to promoter P/A1/04/03 (ref. 17) was inserted into the multiple cloning site of the lambdaattP plasmid pLDR9 (ref. 18). The origin of replication and the kanamycin resistance gene were cut out and the remaining lambdaattP-pIII nonreplicative DNA was religated. Escherichia coli DH5alpha containing plasmid pLDR8 (ref. 18) were transformed with the lambdaattP-pIII nonreplicative DNA and plated on agar plates containing 25 mug/ml ampicillin. After incubation overnight at 42°C, the colonies obtained were replica-plated and ampicillin-resistant clones were identified, named E. coli DH5alpha/pIII. They were subsequently transformed with M13KO7DeltapIII, resulting in the packaging cell line E. coli DH5alpha/pIII [M13KO7DeltapIII].

Production of phage.

To obtain M13KO7DeltapIII helper phage, M13KO7DeltapIII DNA was electrotransfected into E. coli K802 containing pNDI (ref. 15). To obtain hyperphage, M13KO7DeltapIII DNA was electrotransfected into E. coli DH5alpha/pIII. Phage were produced by cultivation in the presence of 0.5 mM isopropyl-beta-D-thiogalactoside (IPTG) at 30°C for 24 h with shaking at 200 r.p.m. Escherichia coli Top10F' or E. coli Xl1blue carrying the pSEXphOx(Yol) phagemid or a scFv library in pSEX81, respectively, were infected with a helper phage preparation at a OD600 of 0.1 at a multiplicity of infection of 20 as described.

Phage quantification by ELISA.

Dilution series of phage were coated in 100 mM NaHCO3, pH 8.6 in MaxiSorb ELISA plates (Life Technologies, Karsruhe, Germany) for 16 h at 4°C. Blocking was done with 2% skim milk powder in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, pH 7.3) for 2 h at room temperature. Phage were detected with the monoclonal mouse antibody B62-FE2 (Progen, Heidelberg, Germany) specifically binding an epitope on the pVIII major coat protein of the M13 phage. M13KO7 phage of known colony-forming units were used for standardization.

Immunoblot.

Approximately 1010 phage were applied per lane on a 10% polyacrylamide gel. Blocking was done with 2% skim milk powder in PBS for 2 h at room temperature. Immunostaining was done with the mouse mAb anti-g3p (MoBiTec, Göttingen, Germany) recognizing the pIII coat protein of M13KO7, visualized with 3,3', 5,5'-tetramethylbenzidene (TMB) substrate (Promega, Madison, WI).

Antigen-binding phage ELISA.

Dilution series of BSA-phOx antigen in 100 mM NaHCO3, pH 8.6, were coated in MaxiSorb ELISA plates (Life Technologies) for 16 h at 4°C. After blocking of unspecific binding with 2% skim milk powder in PBS for 2 h at room temperature, 2 times 109 single-chain phage diluted in 2% skim milk powder in PBS were applied to each well for 1 h at room temperature. After washing six times with 400 mul PBS per well, the bound phage were detected with mAb B62-FE2 as described above.

Panning.

A human scFv fragment library in pSEX81 with a calculated maximal complexity of 2 times 107 was generated from peripheral lymphocyte preparations as described21. Tetanus toxin obtained from Virotech (Rüsselsheim, Germany) was coated to six ELISA wells (Life Technologies) at a concentration of 0.1 mug/ml in 100 mM NaHCO3, pH 8.6, for 16 h at 4°C. The wells were blocked with 2% skim milk in PBS for 2 h at room temperature, after which 1011 phage of the library were applied to each well and incubated at 4°C overnight. After five washes with PBS/0.1% Tween and five with PBS, bound phage were eluted with 1 mug/ml trypsin (Life Technologies) in PBS for 15 min at room temperature. Eluted phage were used for the infection of 20 ml of E. coli XL1blue at a OD600 of 0.4. Infected bacteria were grown on Luria–Bertani agar plates containing glucose and ampicillin, scratched from the plates, and used for the production of antibody phage for the next round of panning as previously described.

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Looks great Devashish Das. I'll wait until I'm sure of not getting a better answer, but I think that paper pretty much covered it. –  J. Musser Jul 14 at 22:08
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I appreciate that. –  Devashish Das Jul 15 at 18:32
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+1 for tackling such a difficult question! –  shigeta Jul 16 at 3:53
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