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PharmOptima is a proud member of Genesis Drug Discovery & Development (GD3), a fully integrated CRO providing services to support drug discovery programs of our clients from target discovery through IND filing and managing Phase I-III clinical trials.
Learn more about GD3SERVICES
PharmOptima scientists have provided biochemistry services to companies world-wide. We specialize in developing custom assays to meet specific client research needs.
What we can do for you:
- cell-based assays
- biomarker assays
- ELISA
- MSD electrochemiluminescence
- ligand binding assays
- Nuclease Protection Assay
- Protein purification and characterization:
- Affinity protein purification methods limited to:
- His-tag
- GST-tag
- MYC-tag
- FLAG-tag
- Fc fusion proteins
- Antibodies via protein A/G
- Traditional methods
- Affinity protein purification methods limited to:
- antibody purification and labeling (eg Biotin and sulfo-tag ruthenium for ECL applications)
- stable mammalian cell line generation and characterization
- protein expression
- eukaryotic expression systems (mammalian cell and insect cell/baculovirus)
MSD and ELISA
Utilizing MSD and ELISA Technology*, biomarkers are available from human, rat, mouse and non-human primate in many cases. A few examples include:
- metabolic markers
- oncology markers
- vascular markers
- cytokines and chemokines
- cell signaling pathways
- Alzheimer’s (Aβ38, Aβ40, Aβ42 and Tau, Total Tau)
- kidney injury
- cardiac and muscle
- liver injury
- inflammation
Custom Assay Development
We provide custom assay development services to customers worldwide. PharmOptima’s scientists can work with you to develop assays to meet your specific research needs. Our scientists have extensive experience in the different custom assay development services listed below.
- Immunosorbant assays, traditional ELISA and ECL
- Development of multiplex immunoassays using MesoScale Discovery technology
- Cell-based assays: signal transduction (eg cAMP)
- Ligand binding assays
- Enzyme assays and kinetics
- Inhibitor mechanism and validation
- MSD Certification such as Human Aß42 and Human Total Tau
What is RNA analysis by nuclease protection?
A nuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence even at low total concentration.
Oligonucleotides are traditionally used in polymerase chain reaction applications for in vitro gene amplification. However, recently, oligonucleotides are being evaluated for therapeutic applications either by increasing gene expression via splicing intervention (e.g., Nusinersin in Spinal Muscular Atrophy treatment), decreasing gene expression using anti-sense sequences to inhibit transcription or translation (e.g., Mipomersin inhibition of apoB-100 translation in the treatment of Familial Hypercholesterolemia) or in the case of aptamers, inhibiting protein activity (e.g., Pegaptinib inhibition of vascular endothelial growth factor, VEGF, in the treatment of macular degeneration).
Pharmacokinetic studies for oligonucleotide drugs require specific assays. One assay format that has been put to use by PharmOptima’s scientists is the nuclease protection assay (Figure 1). The nuclease protection assay (see Figure) uses a labeled nucleotide sequence complementary to the oligonucleotide drug to capture free drug from solution. Following capture, the binding reaction is treated with S1 single stranded nuclease to remove non-bound capture oligonucleotide, removing non-specific signal.
PharmOptima uses Meso Scale Discovery (MSD) technology for this assay. The MSD platform, while similar to ELISA, uses electrochemiluminescence detection to analyze complex sample matrices for a variety of analytes.
Assay Characteristics
- Sensitivity pg/mL
- Large dynamic range pg/mL through microgram/mL
- Assay reproducibility
- Quick turn around time (2-3 hours) once assay is established
- Minimal matrix effects
The nuclease protection assay uses a labeled nucleotide sequence complementary to the oligonucleotide drug to capture the oligonucleotide drug from solution forming a double stranded sequence. S1 nuclease is an enzyme that degrades single stranded DNA and RNA. In the nuclease protection assay, following the capture step, S1 nuclease is used to degrade any non-double stranded nucleic acid leaving the double stranded captured drug “protected”. Signal from the double stranded protected sequence is then used for detection and quantification of the oligonucleotide drug.
Our Spinal Muscular Atrophy (SMA) services begin with research and concludes with a high level of technical expertise.
GD3 researchers have worked with organizations such as the Spinal Muscular Atrophy Foundation, universities and pharmaceutical companies on critical SMA research. SMA, the leading genetic cause of infant death, is caused by defects in the Survival Motor Neuron 1 (SMN1) gene that encodes SMN protein. SMA patients have at least one copy of a similar gene (SMN2) that produces SMN protein, although in reduced amounts. Infantile-onset spinal muscular atrophy is the most common genetic cause of infant mortality, typically resulting in death preceding age two. Low-level production of survival motor neuron protein (SMN) results in a loss of specialized nerve cells called motor neurons that control muscle movement.
In recent years, new SMN-enhancing therapeutics have been developed. Since limited knowledge of baseline and drug-induced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments, clinical trials in this population require understanding disease progression and identifying meaningful biomarkers to hasten therapeutic development and predict outcomes. Clinical studies require a readily accessible means of tracking SMN levels in the patient. GD3 scientists have developed an immunoassay system that monitors SMN protein levels in whole blood. The assay can detect and quantify SMN protein from as little as 5 microliters of whole blood, which can be obtained from a finger prick.
Novel Method Development and Standard use:
- Immuno Assays
- Immunogenicity Assays
- Cell-based Assays
- Nuclease Protection Assays
- Ligand Binding Assays
- Oligonucleotide Assays
- ELISA Assays
- SMA transgenic mouse colony capabilities
- LC-Mass Spec analysis of small molecules
GD3 experts have supported research in published studies of spinal muscular atrophy, including:
- Whole blood survival motor neuron protein levels correlate with severity of denervation in spinal muscular atrophy
- Natural history of infantile-onset spinal muscular atrophy
- Age-dependent SMN expression in disease-relevant tissue and implications for SMA treatment
- Mild SMN missense alleles are only functional in the presence of SMN2 in mammals
- Evaluation of potential effects of Plastin 3 overexpression and low-dose SMN-antisense oligonucleotides on putative biomarkers in spinal muscular atrophy mice (plastin 3, also known as T-plastin or fimbrin)
- Normalization of Patient-Identified Plasma Biomarkers in SMN2 Mice following Postnatal SMN Restoration
Let GD3 collaborative biology/bioanalytical teams generate high-quality leads to advance your programs.