Details

Autor(en) / Beteiligte

• Frisch, Christian
• Knappik, Achim
• Choidas, Melanie
• Tesar, Michael

Titel

Titration of Infective and Noninfective Ff Filamentous Bacteriophages Using a Monoclonal Antibody against g3p

Ist Teil von

• BioTechniques, 2000-07, Vol.29 (1), p.26-30

Ort / Verlag

Natick, MA: Future Science Ltd

Erscheinungsjahr

2000

Quelle

MEDLINE

Beschreibungen/Notizen

• Phage display technology is now widely used as a rapid and powerful approach for identifying and optimizing binding antibodies, peptides and other biomolecules. Phage display selection and screening can be performed in microplates and is therefore amenable to automation. However, one time-consuming step is the determination of phage titers, which is necessary before and during panning. Titration of Ff filamentous bacteriophages is usually done by the determination of plaque-forming units (pfu) or colony-forming units (cfu), which requires serial dilution infection of an E. coli test strain, plating of the cells, overnight growth and subsequent counting of colonies or plaques. For titration of noninfective phages as used in selectively infective phage (SIP) technology, this method can obviously not be used. Alternative approaches such as ELISA using anti M13 antiserum and spectrophotometry are not very accurate; it has been shown that a phage population usually contains a high proportion of polyphages (phages of more than one unit length having more than one genome packaged), especially if the widely used truncated versions of minor phage coat protein III (g3p) are expressed. Therefore, we aimed to develop a method that accurately determines the phage titer without relying on infectivity and that can be used in high-throughput experiments. Since in all types of phage display systems the concentration of the C-terminal domain of the phage g3p protein is directly correlated with the number of phages, we have established an ELISA method using the commercially available mouse monoclonal anti-g3p antibody, 10C3, which recognizes a linear epitope of the C-terminal domain of g3p. The g3p is present most likely in five copies on every phage, reflecting the fivefold symmetry of the phage coat and the pilus.