Description of testing to be performed: Three antibodies provided by the client, will be assessed independently for steady state sodium current antagonism of heterologous human voltage gated sodium channel (hNav1.7) in the whole cell configuration. Chinese hamster over cells (CHO) or Human embryonic kidney (HEK) cells stably or transiently expressing SCN9A (which encodes for hNav1.7) will be voltage clamped at 22°C in a perfusion chamber for experiments where extracellular antibody-induced changes in channel current magnitude and kinetics will be assessed relative to control conditions. The extracellular solution will contain (in mM) NaCl (150), CaCl2 (2), MgCl2 (1), and HEPES (10) and the intracellular (pipette) solution contained (in mM) CsF (100), EGTA (10), NaCl (20), MgCl2 (3) HEPES (10). All solutions will be balanced osmotically to 300 mOsm (+/- 5 mOsm) and will be pH balanced to 7.4 (+/- 1). The steady-state and reversal of (any) antibody effects on the sodium current will be assessed after 3–5 min of exposure and monitored by a -20 to 0 mV depolarization voltage train at 0.1-1 Hz from a holding potential of −100 to -120 mV. The clients will specify the four testing concentrations for each Antibody, otherwise the investigator will initially evaluate the antibody at 1 M. The remaining test concentrations based on the 1 M potency result will be adjusted (increasing or decreasing). Antagonism of the Nav1.7 current by the antibody vehicle will also be tested upon request by the client. At least four replicates for each test concentration of antibody will determined as long as sufficient quantities of antibody are provided and the test concentration of the antibody is soluble based on visual inspection. In addition, changes in the voltage dependence of the Nav1.7 currents will be assessed using variable membrane potential protocols and be compared in the presence and absence of the antibody. Residual linear leak and capacitance will be subtracted from the analyzed sodium current. Data will be acquired using Molecular Devices Pclamp software and a 200B amplifier. Data will be analyzed using Molecular Devices Clampfit and Wavemetrics IGORpro software. The Northwestern or investigator will provide the client with a final written “Report” describing the experimental methods, results and the investigators interpretation of the results. The study will be complete within 8 calendar months of receipt of all three antibodies.
|Effective start/end date||4/15/17 → 4/14/18|
- TetraGenetics, Inc. (Agmt 5/5/17)
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