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Alomone Labs: Twenty Years of Ion Channel Activity

Alomone Labs was established in 1989, and has since grown to be a leading company specialized in the field of ion channel research tools. The activities of Alomone Labs include the generation of novel and specific ion channel, GPCR and neurotrophic factor antibodies as well as the recombinant production of proteins, such as venomous toxins and neurotrophic factors. We proudly describe the past and current technological progress of the Company and some areas of interest responsible for maintaining highly innovative and high quality products from Alomone Labs.

The Company

A venomous animal injects its prey with a mixture of bioactive compounds in order to provoke immediate paralysis. Generally, the venom contains an array of enzymes and other large proteins, and many smaller, highly disulfide bridged, peptide toxins which bind and modulate surface membrane proteins, located especially in nerves and muscles in order to prevent any fast reaction from the prey. These surface membrane proteins are mostly ion channels, a set of proteins which facilitate electrical currents and therefore fast inter and intra cellular signaling.

Peptide toxins and ion channels are in many ways the core of Alomone Labs’ establishment. More than twenty years ago Alomone Labs started as a venom supplier (see reference 3) and soon realized the tight functional and thematic connection between venomous toxins and ion channels. Therefore, the company gradually moved into the design and production of specific ion channel antibodies (see reference 2).

Over the years, a strong technological platform has been created to enable recombinant production of venomous toxins (generally termed ion channel modulators) and growth factors, as well as expanding the design and production of specific antibodies against ion channels, and antibodies against growth factors and their receptors. G-Protein Coupled Receptors (GPCRs), 7-transmembrane spanning proteins, have proven to be important in modulating ion channel activity. Recently, Alomone Labs has also greatly expanded its GPCR portfolio. Alomone Labs’ entire platform includes recombinant protein production and the subsequent chromatographic protein purification and analysis; polyclonal and monoclonal antibodies production, including extracellular antibodies (recognizing their target from the outside), which enables detection in living cells, and fluorescently labeled antibodies.

Alomone Labs puts a lot of emphasis and effort in producing high quality products. An in house professional and dedicated team is deeply involved in thoroughly characterizing all marketed products.

Following production, Alomone Labs’ antibodies are characterized by biochemical applications including western blot, immunohistochemistry, immunocytochemistry and FACS analyses. Proteins, including growth factors and ion channel toxins (as well as some of Alomone’s small molecule modulator products), are characterized biochemically by HPLC and Mass-spectroscopy. In addition, their biological activity is assayed by a team of cellular biologists when cell-based, and by electrophysiologists when electrophysiological measurements are required. Cell-based assays at Alomone Labs include calcium imaging, western blot analysis to detect cell signaling protein phosphorylation, neurite outgrowth, proliferation, apoptosis and cellular migration assays. Our electrophysiological assays mainly involve patch clamp to record ion channel currents from model cell lines and primary cultures or heterologously expressed ion channels in mammalian cells and two electrode voltage clamp (TEVC) recordings for ion channel currents heterologously expressed in Xenopus oocytes.

With all these skills, Alomone Labs’ quality control criteria are strict, and a product is marketed only after the confirmation by the set of binding or biological activity assays mentioned above.

Specificity Issues

With each of its antibodies, Alomone Labs includes the negative control antigen so that the specificity of each antibody could be retested by the end user. Recently Alomone Labs has been making the effort to produce, for several ion channels, a second primary antibody targeted against a different epitope. Hence, similar binding patterns of the two different antibodies will unequivocally prove the specificity and reliability of each antibody.

Specific gene knockout mice are often used as a “gold standard” to confirm the specific interaction of an antibody with its gene product, which is often lost in such mice. Indeed many of Alomone Labs’ antibodies were found specific using this method (see a few examples for Kir3.2P2X4TRPV4HCN1KCa1.1, and Cav1.2 channels in references 4 and 6-10). However, such a gene inactivation approach is not always the ultimate way of demonstrating antibody specificity, since in some cases the gene in question is not knocked out in a complete manner. The P2X7 purinergic receptor-channel is a good example: after years of questioning the specificity of Alomone Labs P2X7 Receptor antibodies, based on their staining of knockout mice tissues (one targeted against an intracellular and one to an extracellular domain of the protein, see reference 1), it was recently published, that P2X7 -/- available mouse strains still express a functional splice variant of the channel, which is recognized by some P2X7 specific antibodies (see reference 5).

Other Activities

Most of the Company’s activity lies in developing and commercializing new products within our existing line of antibodies to ion channels, GPCRs, growth factors and their receptors and proteins including ion channel modulators (toxins) and growth factors. With such a technological platform at hand, Alomone Labs has recently started to explore diagnostic and therapeutic venues related to ion channels. One such activity is our search for ion channels up-regulated in colon cancer to serve as diagnostic markers in colon cancer patients. This activity is within the scope of an Israeli industry-academia consortium that aims to combine novel diagnostic tools with a miniature camera. In this project the company is also developing specific extracellular monoclonal antibodies that will serve as specific markers for infected tissue within the colon.

References
1. Anderson, C.M. and Nedergaard, M. (2006) Trends Neurosci29, 257.
2. Attali, B. et al. (1997) J. Neurosci17, 8234.
3. Garcia-Calvo, M. et al. (1991) Biochemistry 30, 11157.
4. Marker, C.L. et al. (2002) NeuroReport 13, 2509.
5. Nicke, A. et al. (2009) J. Biol. Chem284, 25813.
6. Nolan, M.F. et al. (2003) Cell 115, 551.
7. Sim, J.A. et al. (2006) J. Neurosci26, 9006.
8. Werner, M.E. et al. (2007) Am. J. Physiol.  292, R616.
9. White, J.A. et al. (2008) Learn. Mem15, 1.
10. Zhang, D.X. et al. (2009) Hypertension 53, 532.