Basic screening assays

A useful sensitive primary screening process for antibodies on the basis of their binding to immobilized antigen is the phage EUSA. Individual colonies generated by the selection process can be picked into 96-well format and phagemid rescue carried out. The resultant phage particles can then be used directly in an 

Rescue of phagemid clones in 96-well format for use in ELISA assay 

Pid individual colonies with a sterile toothpick or pipette tip. Transfer the toothpick into 100 pi of 2TYGA in a 96-w ll microtitre plate and grow with shaking (100 r.p.m.) overnight at 30 °C. This is the master plate. 

Prepare a replica 96-well microtitre plate with 100 pi of 2TYGA in each well. Inoculate the replica plate from the master plate. Add 50 pi of 50% cerol to each well of the master plate and store at -70 C. Grow the replica plate with shaking (100 r.p.m.) at 37 °C for approx. 5 h, or until the broth becomes turbid. 

Add 10 pi of M13K07 helper phage in 2TYGA (at 5 X 10 p.f.a/ml, which should give a m.o.i. of approx. 10) to each well of the replica plate. Incubate the plate at 37 C for 30 min without shaking, and then for 30 min with shaking (100 r.p.m.) at 37 C to allow superinfection of the helper phage. 

Prepare a replica 96-welI microtitre plate with 100 p.1 of 2TYCA in each well 

Inoculate the replica plate from the master plate. Add SO of 50% glycerol to each 

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Certainly biomolecular NMR is not the single method which is important for hit identification in pharmaceutical research. It is always a combination of techniques and a team effort that leads to a successful drug. This can involve biologists (basic understanding, assay development, bio-informatics), chemists (both bench chemists and modelers), screening specialists (HTS/natural products) and spectroscopists (X-ray, optical methods, surface plasmon resonance, NMR). 

Table 2 Higher-throughput basic screens (level 1) and lower-throughput toxicology assays (level 2)...

Four basic approaches are conducted for plant selection for antiviral screening assays 1- Random collection of plants followed by mass screening. 2- Ethnomedical approach. 3- Literature-based follow up of the existing leads. 4- Chemotaxonomic approach [12]. The second and third approaches are the most favored ones because of their cost-effective applicability. The selection based on folkloric use proved five times higher percentage of active leads than other approaches. The random approach usually affords more novel compounds with antiviral activity. Combining ethnomedical, phytochemical and taxonomical approaches is considered the best compromise. 

The development of protein chip assays to determine protein function using purified components is a rapidly advancing area. Automated systems for the assay of protein function on chips in parallel for thousands of proteins simultaneously will likely be available in the next few years. These miniaturized arrays will be useful for basic research as well as for diagnostics and drug development. For instance, the combination of protein chips with combinatorial chemistry will allow the simultaneous screening of vast collections of small molecules against vast collections of potential target proteins. 

Not many nonpeptide agonists are yet available, but such compounds have been described — for example, in the angiotensin, CCK, and opioid receptor systems. In fact, for a few receptors, such as the somatostatin, ghrelin, and complement C5A receptors, basically all compounds found by screening using binding assays are agonists. In contrast, for the majority of receptors for which... 

Current efforts favor tumor cell line tests, conducted by the National Cancer Institute (NCI) drug development program [62]. In the current NCI anticancer screen, each compound is tested against 60 human tumor cell lines derived from several cancer types (lung, colon, melanoma, kidney, breast, ovary, brain, leukemia). The tumor cells are seeded on 96-well microtiter plates and pre-incubated for 24 h. The test agents are then added to the wells (five 10-fold dilutions 0.01 -100 pmol/1) and are incubated for 48 h with the tumor cell lines. At the termination of the assay, the cells are fixed in situ with trichloroacetic acid (TCA), washed and dried. Sulforhodamine B (SRB), a dye that binds to the basic amino... 

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