Affinity screening

Once phage or scFv which specifically bind the antigen of interest have been identified it may be necessary to cany out further screening tests to assess which antibodies fiom the positive population have the highest affinities. There are a variety of wtq of achieving this including surface plasmon resonance (BlAcore) screening, fluorescence quench measmement, and competition ELISAs. Descriptions of these protocols are beyond the scope of this chapter. 

Inoculate 10 ml of 2TYGA in a 50 ml Falcon tube with an individual colony of the clone of interest and grow overnight at 30 °C with shaking (200 r.p.m.). 

Split the culture between two 50 ml Falcon tubes and centrifuge at 4000 r.p.m. for 15 min at 4 °C in a pre-chilled Sorvall SS 34 rotor. 

Pour off the supernatant and resuspend the cell pellet in 50 ml of 2TY containing 100 p,g/ml ampidllin and 1 mM isopropyl p-S-thiogalactopyranoside (IPTG) prewarmed to 30 °C. 

Return the cells to a 250 ml flask and grow for 4 h at 30 °C with shaking. 

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Several affinity screening methodologies that include MS-based readout and work under protein-excess conditions have been developed in the past decade [1]. Some examples include affinity selection/mass spectrometry (ASMS Abbott Labs [10]), size exclusion chromatography with LC-ESI-MS (see Chapter 2 and 3 [11-19]), the use of coupled or non-coupled pulsed ultra-filtration/mass spectrometry (summarized in this chapter [11, 20-23]), restricted access phase chromatography (see Chapter 5 [24, 25]), capillary electrophoresis [26, 27], target shift mass spectrometry [28], and multitarget affinity/specificity screening (MASS, see Chapter 10 [29, 30]). 

Ultra-high Throughput Filtration-based Affinity Screening as a Discovery Tool 163... 

We have developed a high throughput ultrafiltration affinity screening method coupled to MS (affinity selection/mass spectrometry ASMS), which works with any soluble target and small molecule library (including natural products). ASMS is amenable to parallelization, efficient and robust enough to allow study... 

Additional Affinity Screening Methodology That Includes Mass Spectrometry-based Readout 177... 

Pulsed ultrafiltration MS (PUF-MS) represents an inline high throughput affinity screening method with a variety of potential uses in the discovery and development of pharmaceuticals [22]. The in-line combination of solution-phase equilibration, ultrafiltration, and electrospray liquid chromatography mass spectrometry (LC-ESI-MS) facilitates the identification of high affinity target-specific... 

Sannes-Lowery, K. A., Drader, J. J., Griffey, R. H., Hofstadler, S. A. Eourier transform ion cyclotron resonance mass spectrometry as a high throughput affinity screen to identify RNA-binding ligands. Trends Anal Chem 2000, 19, 481 91. 

Khasawneh MA, Vallano PT, Remcho VT. Affinity screening by packed capillary high performance liquid chromatography using molecular imprinted sorhents. II. Covalent imprinted polymers. J Chromatogr A 2001 922 87-97. 

Surface Plasmon Resonance New Biointerface Designs and High-Throughput Affinity Screening... 

One potential drawback to multi-target screens is that they are unable to distinguish between different modes of inhibition, so are equally likely to identify substrate binders and enzyme binders. The latter are specifically needed to help unravel enzymatic mechanism. Affinity screens can be used to identify compounds that bind to the enzyme. This type of screen has been successfully used to identify inhibitors of MurF that were subsequently used to obtain a crystal structure to aid in drug design [180,181]. A slightly different approach is a displacement screen, which selects for inhibitors that compete with the substrate for binding. This method has been used successfully to identify small-molecule inhibitors of MurG [182,183]. 

Cyclic nucleotide phosphodiesterase 4 (PDE4) A combination of low affinity screening and high-throughput X-ray analysis led to the identification of a pyrazole carboxylic ester scaffold. The design and in silica evaluation of small libraries exploring three substitution sites led to a 4,000-fold improvement in potency after only two rounds of synthesis. 169... 

Improvement in LC-MS and LC-MS/MS analysis throughput has been reported by the use of monolithic silica columns to increase the speed of chromatography separation [156-158]. The substimtion of an SFC front end to MS in lieu of HPLC has been a growing trend in compound library analysis. It is possible that the use of SFC-MS will be extended to in vitro and in vivo evaluation of library compounds such as ADME and DMPK. CE-MS has not been widely used in the analysis of combinatorial libraries. To date, the application of CE-MS has been frequently in the analysis of mixture-component libraries derived from split synthesis and in the affinity screening of libraries through ACE. However, with the miniaturization of biological screening in the lab-on-a-chip format, CE-MS may find renewed interest... 

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