Automated patchclamp methods for the herg cardiac potassium. The herg fluorescence polarization assay kit provides a set of validated components to perform herg channel biochemical binding studies in the absence of radioligand. The robustness of auroras ion channel screening assays is shown in the figures below. Optimization of the assay conditions buffers, incubation time, and assay plate has provided a highthroughput assay for the rapid determination of herg channel affinity. We are proud to have been contracted by the fda to provide ion channel screening services in support of the cipa intiative. Automated or conventional manual patch clamp screening and concentrationresponse ic 50 assays in panels of cardiac ion channels provide a rapid, comprehensive evaluation of multiple ion channel effects mice for greater insight into potential cardiac risk and more effective decisionmaking in early preclinical development. Whether you want to explore an ion channel as a potential new target or understand cardiovascular liability, or other liabilities, apconix can help you with our electrophysiology expertise, customerfocused flexibility and rapid delivery of high data quality. Hts, single concentration dose response studies, selectivity profiling.
Ion channel assays are performed manually by patch clamp or by qpatch. Normalised concentrationresponse curve of antagonist cisapride against the herg current activated by. The polarization data are shown in figure 2, with the data summarized and compared with patch clamp or radioligand displacement data that have been previously reported in table 1. The predictor herg fluorescence polarization assay kit provides. Korea supplier of cytopatch, auto patch, patch clamp, ion channel, herg assay, instant cells. Why are there current fluctuations in herg patch clamp assay using port a patch nanion.
Reagent kit portapatch standard, cryo box with 81 vials, incuding 27x. Special note for comparison to electrophysiology data. A new homogeneous highthroughput screening assay for. Manual patch clamp evaluation of herg channel pharmacology. Comparison of signal dynamic range between the flipr potassium assay kit and a competitor kit. Recording system for the herg qpatch htx assay the qpatch htx is a highthroughput and automated electrophysiology platform. This application note explains how the flipr potassium assay kit can be run on the flexstation 3. Biochemical herg assays thermo fisher scientific us. In all our tests, including assays of cardiotoxic compounds not correctly identified by the herg assay, reprocardio has generated results in complete agreement with clinical findings.
Completed herg channel pharmacology using 12 cipatraining drugs and manual patch clamp rigs. The comprehensive in vitro proarrhythmia assay cipa. We support client medicinal chemistry optimisation programmes, cipa compliant cardiac safety profiling assays, neuroscience assays and translational assays. Jul 24, 2019 the assay guidance manual agm is a free, bestpractices online resource devoted to the successful development of robust, earlystage drug discovery assays the manual was originally developed by eli lilly and company to provide stepbystep guidance based on tribal knowledge from drug developers for planning and creating projects for highthroughput screening, lead optimization and. Fluxion biosciences ionflux automated patch clamp systems. Although manual patch clamp testing is the gold standard for this purpose, biochemical herg assay allows checking multiple compounds at the earliest stages of drug discovery. With the largest functional kinase activity assay panel in the industry, rbc provides kinase, epigenetic, and other profiling services to over companies worldwide. The comprehensive in vitro proarrhythmia assay cipa guide. Building on more than a decade of own research and a wealth of expertise in the area of multiplex immunoassays, we design, establish and validate miniaturized assays for a multitude of applications such as pkpd studies, druginduced organ injury, pathogen infections and proteinprotein interactions.
This application describes the utility of a new potassium assay kit on flipr tetra high throughput cellular screening system to investigate effects of compounds on herg channel assay activity. Fullyvalidated cell lines for use in cellbased functional assays including manual and automated patch clamp in fluorescent and luminescent platforms. Conventional patch clamping, now also referred to as manual patch clamp to distinguish it from the recently developed automated patch clamp described below see section vi, is accomplished by sealing the small tip of a pipette to the surface of the cell membrane in such a way that is possible to isolate a tiny membrane area patch from the rest of the membrane and to control its. Wholecell configuration of the patchclamp technique in the. Reprocardio 2 human ipscderived cardiomyocytes have been extensively characterized for their functional responsiveness to the known cardiotoxic compounds. Early identification of herg liability in drug discovery programs by automated patch clamp. The results from herg assays have been reported to differ from clinical observations by approximately 30% kramer et al. Applied strict data quality criteria and compared results generated by 3 electrophysiologists using different styles of manual patch clamp rigs. The assay is based upon the principle of fluorescence polarization fp, in which a redshifted fluorescent tracer is displaced from the herg channel by compounds that bind to the channel. Early identification of herg liability in drug discovery. Glp herg is provided through our expert alliance partner, physiostim visit the european centre of excellence page for more information. Predictor herg assay test kits were obtained from invitrogen.
Ionic current measurements request a study outline from ipst wholecell current amplitude and kinetics measurements verify the results of the interaction of a test article with a selected ionic current. Metrion biosciences offers nonglp herg screening services using the qpatch automated gigaseal patch clamp platform. Flipr assays are a set of flurometric assay technologies which is compatible for fliprtetra. Because of its high data quality, robust recordings and superior level of automation, the patchliner is an exceptional tool routinely used by the pharmaceutical industry, cros and academic institutions. Ion channel targets remain a top priority for many pharma and biotechs with most looking to increase their screening efforts in 2006. Analysis of a new high throughput screening detection. Guanosine 3, 5cyclic monophosphate cgmp is formed via the action of guanylate cyclase on gtp. The qpatch performs up to 48 independent patch clamp experiments in parallel in a 48channel chip array with microfluidic chambers qplate. Precompound current and postcompound current are measured by patch clamp, and applied to the calculation of herg inhibition.
Why are there current fluctuations in herg patch clamp assay. The impact of automated patch clamping apc on ion channel screening is now evident, particularly in early noncompliant herg liability testing, where apc is rapidly becoming the new gold standard technology. The human etheragogo related gene herg encodes the inward rectifying. Therapeuticdisease area build your own assay tools assay ready kits. The assessment of electrophysiological activity in human. The popular herg assay is an example of ionic current assay, saimed specifically at the wellknown herg channel. Highthroughput electrophysiological assays for voltage gated. Evaluation of the rubidium efflux assay for preclinical identification of herg blockade. Competitive binding assay of 10 known herg blockers using the predictor herg fluorescence polarization assay. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Acroscell experienced technicians allow us to provide customized assays. Advanced microfluidics automated patch clamp system with continuous flow and temperature control.
Combined with nanions perfusion system it can be operated either manually. Why are there current fluctuations in herg patch clamp. The portapatch is a miniaturized patch clamp system supporting gigaseal recordings. A single ion channel conducts around 10 million ions per second. Recent advances in automated patch clamp technology apc have begun to show promise of becoming an alternative compliant test to the gold standard manual method. Jun 04, 2015 an oxford university study that used manual patch clamp to simulate changes in the action potential duration concluded that this is a better approach than considering herg block alone.
Normalised concentrationresponse curve of antagonist cisapride against the herg current activated by voltage. The cyclic amp xp assay kit is a competition enzymelinked immunoassay used to determine camp levels in cells or tissues of interest. Predictor herg fluorescence polarization assay kit. Guidances are available regarding the herg assay and can be found through the fda. The comprehensive in vitro proarrhythmia assay cipa initiative was established to develop a new paradigm for assessing proarrhythmic risk, building on the emergence of new technologies and an expanded understanding of torsadogenic mechanisms beyond herg block.
The predictor herg fluorescence polarization assay kit provides a set of validated components to perform herg channel biochemical binding studies in the absence of radioligands. Typically, potential drug compounds are tested using a patch clamp electrophysiology test. Ionflux mercury complements manual patch clamp systems by delivering plate reader simplicity to complicated ion channel assays. Here we propose a drug screening strategy that combines manual patch clamp technique with a specially designed drug cocktail application algorithm. Diverse coverage over 60 targets, from voltagegated channels to slow and fast. Protocol details and the translation of criteria known from manual patch. Dofetilide displacement binding assay membrane aliquots from herg transfected hek293 cells were thawed, homogenized, and diluted in 50 m m.
In manual patch clamp recordings of transfected cho cells, grepafloxacin inhibited herg channel activity with a potency of 50100. The predictor herg fluorescence polarization assay kit provides a set of validated components to perform. However, the traditional manual patch clamp assay has very limited throughputusually one compound per day per individual researcher. In this assay, cho herg cells or hek293 herg cells are used to specifically assess the effect of test compounds on the herg channel. In addition to confirming the activity of potential hits from high or medium throughput screens, manual patchclamping can be used to assess mechanism of action of compounds and to determine the effects of compounds on the.
Patch clamp technique is the gold standard for carrying. Identification of human etheragogo related gene modulators. The cayman chemical cgmp assay is a competitive elisa that can be used for quantification of cgmp directly obtained from cell lysates, tissue homogenates, plasma, or urine. A manual patchclamp electrophysiology platform is the gold standard method used for reporting glp compliant herg safety testing as part of an investigational new drug ind submission. The use of manual patch clamp recording in drug discovery is limited by ultralow throughput. Pdf early identification of herg liability in drug. This method is considered comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. No matter the stage of drug discovery, obtaining quick and accurate results is key to any research assay. Wholecell configuration of the patch clamp technique in the herg channel assay to predict the ability of a compound to prolong qt interval. I am doing herg voltage clamp protocol using port a patch but i am unable to get a steady current. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels. Dec 14, 2015 however, with good assay optimization, an acceptable correlation between compound potencies derived from ionworks assays and manual patch clamp electrophysiology can be achieved 36,39. Evaluation of the rubidium efflux assay for preclinical.
Patch clamp technique is the gold standard for carrying out the herg assay. Metrion also offers nonglp herg screening services on the manual patch clamp platform. High throughput ion channel gaba receptors, nmda receptors, nicotinic receptors, cipa assays at manual patch clamp cost. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. Many factors can impact data quality hence reproducibility. Characterization of herg channel blockers using the flipr.
Crosssite and crossplatform variability of automated. Manual patch clamp assay manual patch clamping is the goldstandard for the investigation of ion channel activity. Admetox, biochemical assays analysis of a new high. A manual patchclamp electrophysiology platform is the gold standard method used for reporting. It is a regulatory requirement that an assessment of herg inhibition should be made by manual patch clamp to good laboratory practise glp standards. Performance of the predictor fluorescence polarization assay. Manual patchclamp recording for mediumthroughput ion. Automated patch clamp assays have been successfully used in a.
To address this need, the predictor tm herg fluorescence polarization assay kit from invitrogen has been developed. By optimizing various cell patching parameters and a twostep voltage protocol, we developed a high throughput apc assay for the voltagegated sodium channel nav1. This membrane prep is derived from a herg stable cell line for lotto lot. Identify potential liability issues in early stages of drug. Screening compounds for hazardous side effects on herg channel early in the drug development process is likely to cut the drug development costs. Aid 307664 inhibition of herg current at 1 um by patch. While the patch clamp method works very well for identifying compounds that block potassium ion flow through herg channels, it requires a high resistance seal be made. The control tail current amplitude from a patch clamp assay is analogous to the negative control value in the predictor herg fp assay. An alternative to either manual or automated electrophysiology is a functional assay that. With our portfolio of capabilities, we can support screening of compounds from discovery through ind enabling studies. In addition to confirming the activity of potential hits from high or medium throughput screens, manual patchclamping can be used to assess mechanism of action of compounds and to determine the effects of compounds on the biophysical properties of a channel.
The patchliner is a fully automated patch clamp platform offering medium throughput and vast experimental freedom. Early identification of herg liability in drug discovery programs by. Metrion biosciences ion channel screening integrated. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium. Each kit contains enough reagents for 400 wells in a 384 well format. Therefore, unless very low concentrations of test compounds are included in the compound titrations, the full assay window will not be. Figure 2 main assay technologies used to study ion channels targets in drug discovery. Manual wholecell patchclamping of the herg cardiac k channel. Conventional manual patch clamp evaluation of herg potency in the presence of 100% fetal bovine serum fbs, dialyzed evaluates the effect of drugprotein binding on herg inhibition. Typically, herg potencies measured by automated and manual. The high cost of automated patch clamp drug screening makes it unavailable for the majority of electrophysiological laboratories. This article describes protocols for automated electrophysiology screening of compound effects on the herg channel current.
However a major limitation started to appear in many hts systems studying herg and potassium channels in general is the lack of assay stability and longevity compared to manual patch clamp studies. That technique required a huge effort, training, luckiness and patience, but good results acceptable for. I am using herg hek cells for drug screening using automated electrophysiology system. The predictor herg fluorescence polarization assay kit provides a set of validated components to perform herg channel biochemical binding studies in the absence of radioligand.
The patch clamp technique allows the investigation of a small set or even single ion channels. Sample traces recorded from qpatch automatic patch clamp system a, manual patch clamp b and mea system c. Automated patch clamp an overview sciencedirect topics. Patch clamp electrophysiology remains the gold standard for in vitro herg measurement, whereas the ecg or ekg is the main in vivo approach. The impact of automated patch clamping apc on ion channel screening is now. Invitrogens predictor herg fluorescence polarization assay. Axon instruments patch clamp systems provide the complete workflow solution for patch clamp electrophysiology that is used for whole cell patch clamp, extracellular, and singlechannel recordings. Predictor herg fluorescence polarization assay frequently. Cold flux assays are also available for limited compounds or candidates that cannot be screened with manual patch clamp, with a strong linear correlation r0. Patch clamp herg safety testing services herg human etheragogorelated gene, in italics, or kcnh2 in the new nomenclature is a gene that encodes the poreforming alpha subunit of a voltagegated potassium channel expressed in the heart and in nervous tissue. Pluricyte cardiomyocytes action potential recordings using manual patchclamp exhibit a low resting membrane potential 78 mv, fast upstroke velocity and a welldefined action potential plateau demonstrating a relatively high level of maturity. Luminex protein assay services nmi tt pharmaservices. Explore rbcs large variety of electrophysical assay services for drug discovery, including the manual patch clamp assays. Cytopatch the handsfree manual patch clamp platform.
The predictor herg kit is a homogeneous fluorescence assay that has a simple addandread format, supports highthroughput screening, and provides high accuracy, solvent tolerance, and stability over time. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies. Hts screening of herg blockers using 2ndgeneration. In addition, invitrogen also supplies similar assay kits. The cells used in qpatch are the cho herg stable cell line, while in manual patch clamp are hekcav1. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patch clamp electrophysiology studies. Performance of the predictor fluorescence polarization. The correlation between data obtained with automated patch clamp qpatch 16 and manual patch clamp was high. Ic50 of flecainide for iherg in cho cells expressing herg channels qpatch and in hek cells expressing herg channels manual patch clamp. Assay guidance manual national center for advancing.
The results are in good agreement with manual patch clamp data crumb et al. High throughput screening technologies for ion channels. The comprehensive in vitro proarrhythmia assay cipa guide sedating antihistamine for symptomatic treatment of allergic rhinitis sneezing, itchy or runny nose, watery eyes. Although the herg assay is a useful screening system in the development of new drugs, the results from this assay do not entirely explain the occurrence of tdp in humans nozaki et al. The binding assay was carried out according to the kit instructions. Inhibition of herg current at 1 um by patch clamp electrophysiology assay. In preclinical drug development, seldane gave no cardiac risk signals no effects on cardiac action potential or electrocardiographic qt interval. However, a number of potential sources of errors in setting up herg channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported. Since manual patch clamping has long been regarded as the gold standard for evaluating herg safety by fda and other regulatory institutions, creative biolabs provides firstclass manual patch clamp electrophysiology measurement of herg inhibition with high accuracy.