Assay phage display

Sompuram SR, Kodela V, Ramanathan H, et al. Synthetic peptides identified from phage-displayed combinatorial libraries as immunodiagnostic assay surrogate quality-control targets. Clin. Chem. 2002 48 410-420. 

The procedure for epitope mapping by the phage display method involves biopanning, amplification, assay for positive colonies, DNA isolation, and sequencing. Some steps of the procedure must be performed at the same time. Therefore, we describe the protocols in Subheadings 3.1. and 3.2. on a day-by-day manner. All steps of biopanning and phage amplification should be carried out under sterile conditions, when possible. 

Unlike phage display and other screening technologies that rely on ligand binding, flow cytometry can be readily used to select clones either on the basis of binding or catalytic activity. Numerous enzymatic assays have already been adopted for use with flow cytometry and suitable commercial probes are available from vendors such as Molecular Probes. 

Examples of such protein-protein interaction selection systems are phage display (Smith, 1985 Winter et al., 1994), display on other viruses (Kasahara et al., 1994), bacterial surface display (Georgiou et al., 1993 Daugherty et al., 1999), yeast display (Kieke et al., 1997 Boder and Wittrup, 1997), the yeast two hybrid system (Fields and Song, 1989 Chein et al., 1991), and protein-fragment complementation assays (Pelletier et al., 1998). These methods all contain a necessary in vivo step, which has a number of disadvantages that will be discussed in the following sections. 

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. 

Catalytic antibodies were first reported in 1986 by Lerner and Schultz for the hydrolysis of simple esters. Since then over 100 different reactions have been catalyzed by antibodies, from enantioselective preparative reactions to prodrug activations in vivo [1]. New methods have been developed, including specific immunization protocols, direct screening assays for catalysis, and manipulations with recombinant antibodies via phage display. This article gives an overview of work done towards applying catalytic antibodies for synthetic organic reactions by our and other laboratories. 

A large variety of assays has been adapted to utilise protein microarrays. At its current state, the detection of immobilised antigens with antibodies is still the most common application. Protein and antibody arrays have been used for the selection and characterisation of novel antibodies from phage display libraries and for the identification of antigens (e.g., involved in autoimmune diseases). 

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