Assay Development

GVK BIO offers customised assay development services to help you reach your goals quickly and cost effectively. With a flexibility to adapt assays developed at the client’s site, we take up the task of screening at our facility. Our scientific team with sound technical and analytical skills has developed more than 200 assays against the targets that include GPCRs, Ion channels, transporters and several kinases with a novel methodology. The dedicated facility for radioactive assay readouts enables us to handle assay volumes that range from low to high throughput screening of drug candidates using radiolabelled C, P, H and I reagents.

Increasingly, enzymes are being recognised as potential drug targets in drug discovery and GVK BIO offers high quality assay services with various readouts and formats.

We routinely use biochemical assay technologies, typically including Luminescence, Fluorescence, Fluorescence Polarisation, IMAP, Time Resolved FRET (HTRF), Alpha Screen, SPA, etc. and Cellular Assay Technologies, typically including reporter gene assay, DELFIA, ELISA, CLIA, Alpha Lisa, etc.

Assay Standardisation & Validation Include:

  • Assay development plan
  • Assay validation based on established practice
  • Identification of physical & statistical parameters critical for assay validation
  • Standard Operational Procedures (SOP)
  • Assay run with a set of reference compounds with rank order potency and ascertainment for reproducible dat

Screening & Assay Labs

Our Receptor Biology lab has capabilities to perform receptor binding and functional assays. With the support of our Molecular Biology lab, we generate stable cells expressing target receptors. Our state-of-the-art Cell Culture lab enables us to culture at least 5 billion cells every week. Membranes are prepared from bulk cells, characterised, validated and used in the binding assay for screening of NCEs. Alternately, cells would be frozen for further culturing. We employ filtration or SPA technologies for the binding assays.

Radioligand binding assays are developed for several therapeutic targets. Our targets include various:

  • GPCRs
  • Ion channels
  • Transporters
  • Enzymes

GPCRs

G-Protein Coupled Receptors (GPCRs), also known as 7-transmembrane Receptors are important targets for drugs to treat various disorders. Disruption in their regulation causes inflammatory and respiratory diseases, cancer, cardiovascular diseases and CNS disorders like Alzheimer’s, Parkinson’s disease, etc.

To address the need of the hour, we have established various cell lines expressing GPCRs and validated the assays.

DRC of reference compound using dopamine D2 receptor

DRC of reference compound using dopamine D2L receptor

GPCR Functional Assay

As an extension to our receptor binding assay platform, we have developed in-house functional assays to screen the GPCR targets. Ligand binding to GPCR promotes G-Protein coupling, initiating signal transduction pathways that trigger a series of cellular responses.

At GVK BIO, we have capabilities for measuring GPCR functional responses by developing cAMP assays for Gs coupled receptors and Ca2+ measurements for Gq coupled receptors. We also have capabilities for performing Guanine nucleotide binding assay ([35S] GTPγS assay) for Gi coupled receptors.

cAMP Assay

For GPCRs coupled with Gs G-protein, cAMP assay is employed to determine the functionality of the expressed protein. In GVK BIO we employ cAMPGlo assay and Alpha screen technology to perform cAMP assay.

Ca2+ Release Assay

Ca2+ release assay is used for GPCRs couple with Gq G-protein. A fluorometric assay using FLIPR is employed to determine the Ca2+ release by GPCRs. The Ca2+ released when GPCR is activated by specific ligand binds to a dye and emits fluorescence at specific wavelength.

Radioligand Binding Assays

The principle of radioligand binding assay is based on labelled radioligand molecules binding to specific receptors, transporters, enzymes or any protein of interest. Measuring the rate and extent of competitive binding between the radiolabelled ligand and unlabelled compounds provides us information on the affinity of the compounds to the specific protein.

GTPγS Assay

GPCR occupation by agonists leads to guanine nucleotide exchange on the G protein α-βγ complex; GDP bound to Gα subunits dissociates and is immediately replaced by GTP. GTP-bound Gα subunit then detaches from the Gαβγ complex and the dissociated βγ and Gα-GTP subunits are capable of downstream signalling. Non-hydrolysable analogues of GTP, such as [35S] GTPγS and [Eu] GTPγS allow measurement of GPCR agonist induced GTP incorporation onto Gα subunits. GTPγS incorporation is an index of GPCR activity based on agonist activation and antagonist inhibition.

DRC of carbachol using CHO cell Lines over
expressing muscarinic M3 receptor

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