In Nutshell, These Two Assay Methods Are Very Helpful In High Throughput Screening Technology

Introduction

High throughput screening technology is a useful tool to screen hundreds of samples in a very short time provided a proper assay method has been developed. The development of most accurate, easiest, productive, time and cost effective assay method makes the high throughput screening technology a success story. This post provides the basic concept of principle of two very efficient TNF-alpha screening assays that can be used to screen the samples for hit to lead development.

What is TNF- alpha?

Tumors necrosis factors are of three types, i.e. TNF-alpha, TNF-beta and LT- beta. TNF- alpha is a cytokine involved in inflammatory responses by activated macrophages and some other cells. TNF- alpha is an endogenous pyrogen that has the ability to induce inflammation, prevent viral infection and induce apoptosis. TNF- alpha is involved in a variety of signaling pathways in the cells including apoptotic pathway, NF-kB activation by binding to receptor type I and II (TNFRI, TNFRII). Overproduction of TNF- alpha is related to a variety of inflammatory diseases (Corhns disease) [1]. Inhibitors of TNF- alpha (which are ultimate inflammation and apoptosis inhibitors) can be used to treat the diseases which elevate the levels of TNF- alpha . High throughput screening approach can be used to screen compound libraries against high levels of TNF-alpha . ELISA and radioisotope labeled immunoassays are commonly used for screening analysis. Nevertheless, these processes involve the drawbacks of excessive washing and radioactive wastes. There is a need to simple and beneficial assay which can target the TNF-alpha in an effective manner.

HTRF based TNF- alpha Assay

Homogenous time resolved fluorescence technology is the combination of FRET (fluorescence resonance energy transfer) technology with time resolved fluorescence measurement in order to eliminate the short lived background fluorescence. Following figure explains the principle of assay which involves the production of two types of antibody conjugates. In one of the conjugate the anti TNF-alpha is conjugated with XL665. On excitation with 320nm waves, XL665 produces fluorescence of 615nm even in the absence of TNF-alpha . The second conjugate is made up of anti TNF-alpha with cryptate. In the presence of TNF-alpha in the samples, the two antibodies come in close proximity. This closeness causes in successful fluorescence resonance energy transfer between two flourophores. When such a sample is excited with 320nm waves, emission fluorescence is observed at 665nm. The increase of fluorescence at 665 nm is proportional to the levels of TNF-alpha in the test samples [2]. This assay is easy and reliable for high throughput analysis of large number of samples.



AlphaLISA Based Assay

AlphaLISA is another assay which can be used to target TNF-alpha. This method involves the creation of beads. One of the beads is coated with streptavidin (blue circle) which has the ability to bind with biotin labeled anti TNF- alpha. The other bead is coated with TNF-alpha specific antibody. When a laser beam of 680nm is used for excitation, singlet oxygen is released form the first bead and moves to wards the second bead (anti TNF- alpha coated). As the first bead produces or donates singlet oxygen, it is also called as donor bead while the second bead is usually called as acceptor bead on account of accepting or receiving the singlet oxygen. On receiving the singlet oxygen, the acceptor bead produces fluorescence of 615nm. Again, the extent of fluorescence is directly proportional to the amount of TNF- alpha present in the samples [2].



Conclusion

In nutshell, these two assay methods are very helpful in high throughput screening technology in a quest to discover more hits which can be further optimized to leads. These assays can be quickly and easily performed in 1536-well HTS plates. These methodologies not only add a beneficial assay technique for HTS of TNF-alpha but also open doors for the newer related concepts that can be followed to established to develop even better assay techniques for parp inhibitors, kinase inhibitors, egfr inhibitors etc.!