Phage capture

Phage antibodies are directly used to label tissue sections. Interpretation of the data is performed by a team of experts including histochemists and must be subjected to cross-examination. Images are captured using microscopes with a digital camera attachment and archived in databases for further analysis of pathologists. 

Amplified electrochemical detection of DNA in monolayer assemblies was accomplished by the conjugation of bioelectrocatalytic transformations to the DNA recognition events. This was exemplified with the amplified electrochemical analysis of M13 phage DNA (Fig. 12.20a).75 A capturing nucleic acid, (20), complementary to... 

In this approach, the phage antibodies react with biotinylated antigen in solution, and the complex is then captured using Streptavidin coupled to Dynabeads. 

Take an aliquot of 10 pL for measuring the input phage titer and proceed immediately to the capture with the remaining 990 pL. 

In this strategy, phages are affinity-captured on immobilized substrate under conditions in which the enzyme is inactive, for example, in the absence of an essential metal ion or a cofactor. Active phage-enzymes are then eluted by triggering catalysis by addition of the metal ion or cofactor, taking advantage of the lower affinity for the product than for the substrate. 

As shown in Figure 6.1, the protocol starts with labeling the phages with the substrate. Several approaches have been used for this labeling step [11,13,14]. The active enzymes are then turning over the substrate into product by intraphage catalysis. Finally, the pro duct-labeled phages are selected by classical affinity capture. 

In this strategy, it is very important to avoid interphage catalysis. Therefore, the phage concentration should be kept low (lower than 10-9 M) and phages should not be precipitated with PEG. Because removal of excess label is generally required before affinity capture, it should be done by phage dialysis or size-exclusion chromatography. 

A constant out/in ratio could mean that no clones are being selected. For some strategies that require low-affinity capture for selection, the level of specifically captured phages might always be below the background level. It is therefore worth analyzing the selected phages, because effective enrichment may have occurred. 

The rabbit antibodies used for capturing MAbs may also interact with some phage from the library. This might result in selection of many non-MAb specific clones. Thus, the library should be pre-incubated... 

Release of captured phage carrying catalytic antibodies... 

FIGURE 19.4 Biopanning selection through biotin-streptavidin interaction. The library is incubated with the biotinylated target in solution phages displaying the active binding peptide are captured on a streptavidin-coated Petri dish, eluted, and amplified. 

Fig. 5.1. The principle of phage display is based on in vitro selection of peptides or proteins expressed at the surface of filamentous phages. In a biopanning experiment, specific binders can be captured from a library and their genes can be amplified by infection.

In order to avoid these limitations, efforts have been made to find ways of coupling substrate turnover directly to a selection process. Two groups have devised a method that involves the attachment of a substrate to a phage-enzyme in a way that allows its intraphage interaction with the enzyme. Phage-displaying active enzymes are able to convert the substrate into product. They can be captured from mixtures with product specific reagents or antibodies. 

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