Anti-phage antibodies

Neutralizing anti-phage antibodies have been reported in phage therapy trials in humans (Kucharewicz-Krukowska Slopek, 1987) and animals... 

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

A5. Andrieu, J. M., Mamas, S., and Dray, F., Detection of anti-estradiol antibodies and of 17f)-estradiol by means of a 17fl-estradiol-bacteriophage T4 conjugate. Dependence of the haptenated phage reaction on several physical parameters. Eur. J. Immunol. 4, 417-421 (1974). 

The isolation of disease-related antibodies specific for either melanoma [ 180], the autoimmune thyroid peroxidase antigen [174], or neutrophil antigen characteristic of ulcerative colitis [17] from nonnaive patient repertoire phage libraries has been reviewed above in the Panning on cells section. The isolation of anti-viral antibodies with diagnostic or therapeutic potential from antibody repertoire libraries has been described in earlier papers e.g. against HIV [197], hepatitis B virus [198] and human respiratory syncytial virus [199],... 

Chowdhury PS, Chang K, Pastan I, Isolation of anti-mesothelin antibodies from a phage display library, Mol. Immunol., 34 9-20, 1997. 

Cai X, Garen A, Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells, selection of specific antibodies from single-chain Fv fusion phage libraries, Proc. Natl. Acad. Sci. USA, 92 6537-6541, 19. 

Finnern R, Bye JM, Dolman KM, Zhao MM, Short A, Marks JD, Lockwood MC, Ouwehand WH, Molecular characteristic of anti-self antibody fragments against neutrophil cytoplasmic antigens from human V gene phage display libraries, Clin. Exp. Immunol., 102(3) 566-574, 1995. 

Griffiths AD, Malmqvist M, Marks JD, Bye JM, Embleton MJ, McCafferty J, Baier M, Holliger, KP, Govick BD, Hughes-Jones NC, Human anti-self antibodies with high specificity from phage display libraries, EM BO J., 12(2) 725—734, 1993. 

Shadidi, M. and M. Sioud (2001). An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library. Biochem Biophys Res Commun 280(2) 548-552. 

The presence of the enzyme-g3p fusion protein should also be detected by western blot analysis with an anti-g3P and/or an anti-enzyme antibody. With anti-g3p detection, the relative intensities of the fusion and the free g3p proteins should be in qualitative agreement with the displayed level evaluated by the ratio between the enzyme and the phage concentration. 

In some cases, functional assays have been used to screen phage-display peptide libraries. For example, a phage-display hexapeptide library was constructed with an epitope tag distal (N-terminus) to screen for peptide substrates of a specific protease. All the phages were captured by an immobilized anti-epitope antibody. After incubation with a tissue plasminogen activator (tPA), phages that expressed a peptide substrate for tPA were released for subsequent rounds of selection. A similar approach was applied to discover peptide substrates for HIV-1 protease. 

Malone J, Sullivan MA (1996) Analysis of antibody selection by phage display utilizing anti-phenobarbital antibodies. J Mol Recognit 9 738-745. 

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