Phage neutralization

Infect 1-mL culture of HB2151 with 10 pL (about 105 tu.) of the phage eluted and neutralized at the last round of selection. Plate 0.1, 1, 10, and 100 pL on TYE-AMP-GLU. Incubate overnight. 

A recent XRD study of CL/DNA complexes has revealed that the addition of linear lambda-phage DNA (48, 502 bp, contour length=16.5 pm) (Raedler et al., 1997) or plasmid DNA (Lin et al., 2000) to binary mixtures of cationic liposomes (mean diameter of 70 nm), consisting of mixtures of neutral lipid DOPC and cationic DOTAP, induces a topological transition from liposomes into collapsed condensates in the form of optically birefringent liquid crystalline (LC) globules with sizes on the order of 1pm. 

Release the DNA from the bacterial cells or phage using alkali. Neutralize and hybridize with radiolabeled probe. 

The affinity of antibodies selected from naive, synthetic, or even immune phage libraries is typically sufficient for use as a research reagent, but too low for some specific therapeutic applications such as viral neutralization or tumor imaging. For many applications, affinity maturation can be bypassed completely by the construction of multivalent molecules. If this is not sufficient selected antibody clones may be affinity matured (see Figure 18.13). 

Ames RS, Tornetta MA, Jones CS, Tsui P, Isolation of neutralizing anti-C5a monoclonal antibodies from a filamentous phage monovalent Fab display library, J. Immunol., 152 4572-4581, 1994. 

Allow the filter to remain in contact with the A phage plaques for about 4 min, then carefuUy remove (avoid lifting the top agar) and place the filter, plaque side up, onto Whatman 3MM paper previously saturated with denaturation solution. Remove trapped air bubbles and leave for 4 min then transfer onto Whatman 3MM paper previously saturated with neutralization solution and leave for 4 min. After neutralization, place the filter on dry Whatman 3MM paper and allow it to air-dry. Repeat these steps for each KP used and for each replica filter obtained from the same KP. 

Figure 25. Scheme of Weissmann s experiment [66] for determining error rates of phage Qp. Success critically depends on finding nearly neutral mutant k, which grows up about as quickly as master copy m, i.e., while being replaced by revertant master copy with... 

RS Ames, MA Tometta, LJ McMillan. Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library. J Immunol 1545 6355-6364, 1995. 

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