Cell Biology Assays

1. Target validation

Target validation includes the following stages:
  • Discovering a biomolecule of interest
  • Investigating the expression of the molecular target within the tissue of interest
  • Evaluating its potential as a target to understand if it is directly involved in the disease process
  • Designing a bioassay to measure biological activity to verify if target modulation produces the desired therapeutic effect.
  • Constructing a high-throughput screen.
  • Performing screening to find hits and evaluating the hits
We offer a range of strategies and capabilities for modulating gene expression in vitro. These include but are not limited to the use of antibodies, negative dominant controls, antisense oligonucleotides, ribozymes, and small-interfering RNAs.
Our services to support target validation and discovery:
  • We can offer support and assay design for protein and novel target expression in a range of normal and diseased tissue, including:
    1. IHC and novel marker assay development & co-localisation studies
    2. Phosphorylated marker assay development
  • High-throughput target validation assay that represents biology
  • Molecular level
    1. Screen enzyme inhibitors or activators
  • Cellular Level
    1. Verify the involvement of the protein in the disease state (often use gene silencing siRNAs).
    2. Understand the protein pathways and interactions.

2. Toxicity profiling

Drugs research and development cost every year over $145 billion in US only. One of the key contributors to these rising costs is unacceptable drug safety profile either detected during Phase 1 Clinical Trial (50-60%) or during Phase IV Post marketing surveillance where some severe side effects were unnoticed during the phase I clinical trial. Of note, it has been described that in 30% of the studied cases the animals showed a toxic effect on a different organ compared to human (https://www.gwern.net/docs/dnb/2000-olson.pdf)
Main point of Preclinical testing is to determine the potential drug adverse effects, toxicity and drug-drug interactions before the drug is tested in either animal models or Phase 1 clinical trial. Hepato and renal in vitro toxicity tests are fundamental for pre-screening drug compounds to reduce costs for in vivo testing and Clinical Trials.
Caco2 – Intestinal epithelial cancer cell line was treated with increasing concentrations of Acetylsalicylic acid and Diclofenac sodium for 24 hours. Supernatants were then collected and LDH release was measured. Results are expressed as absorbance (A) or as normalised to the positive control (B) representing 100% lysis. Significance was analysed by one-way ANOVA when compared to no drug treatment ***p<0.001
HeLa cells were seeded at a density of 10,000 cells/well and treated with Staurosporine for 4 hours at concentrations of: 10µM, 5µM, 2µM, 1µM, 500nM, 250nM, and 125nM. The Caspase-Glo 3/7 Reagent was added directly to cells in 96-well plates; the total volume was 80µl per well. The assay was incubated at room temperature for 30 minutes before recording luminescence. Each point represents the average of 3 replicates (error bars represent ±SEM). The blank control value has been subtracted from each point.
Cytotoxic profile of a test antibody (1°Ab) in the presence of 2° antibody conjugated with-MMAE toxin. The viability of Kasumi-3 was analysed using Cell-Titer Glo Luminescence assay. A statistically significant decrease in Kasumi-3 cell viability was observed with concentrations above 0.01 nM test antibody in presence of 2°Ab-MMAE when analysed using One-way ANOVA, followed by Dunnett’s post-hoc multiple comparison test (*p<0.02; **p<0.005; ***p<0.0003, <0.0001; ±SEM).

Kidney Injury Biomarkers

Acute kidney disease Chronic kidney disease Nephrotoxicity
Neutrophil gelatinase-associate lipocalin (NGAL) Asymmetric dimethylarginine (ADMA) N-acetyl-β-D-glucosaminidase (NAG)
interleukin-18 (IL18) Neutrophil gelatinase-associate lipocalin (NGAL) Glutathione S-transferases (GST)
Kidney Injury Molecule-1 (KIM1) Kidney Injury Molecule-1 (KIM-1) Gamma-glutamyltransferase (GGT)
Cystatic C (CST3) Liver-type Fatty acid binding protein (L-FABP) Kidney Injury Molecule-1 (KIM1)
Liver-type Fatty acid binding protein (L-FABP) Lactic dehydrogenase (LDH)
Insulin like growth factor binding protein (IGFBP7)
Tissue metallopeptidase inhibitor 2 (TIMP-2)

Kidney Injury Biomarkers across Species

Human Mouse Rat
Albumin Clusterin Albumin
Clusterin Cystatin C Calbindin
Collagen IV Epithelial growth factor Clusterin
Cystatin C Interferon gamma-induced protein 10 Cystatin C
Glutathione S-transferases Kidney Injury Molecule-1 Glutathione S-transferases
Interferon gamma-induced protein 10 Lipocalin-2 Interferon gamma-induced protein 10
Kidney Injury Molecule-1 Osteopontin Kidney Injury Molecule-1
Lipocalin-2 Renin Lipocalin-2
Liver-type Fatty acid binding protein β-2-Microglobulin Osteopontin
Macrophage activator protein Tissue metallopeptidase inhibitor 2 Tissue metallopeptidase inhibitor 2
Matrix Metalloproteinases (MMP9) Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor
Osteoactivin β-2-Microglobulin (β2M)
Osteopontin Interleukin 2,6,8,10 (IL2, IL 6, IL8, IL10) Interleukin 2,6,8,10 (IL2, IL 6, IL8, IL10)
Renin Trefoil factor 3
Trefoil factor 3
a-1-Microglobulin
Heat Shock Protein 70 (HSP70)
Interleukin 2,6,8,10 (IL2, IL 6, IL8, IL10)

Liver Injury Biomarkers

Human Rats
Liver-Type Arginase 1 (ARG1) Liver-Type Arginase 1 (ARG1)
Malate dehydrogenase 1 (MDH1) aspartate transaminase 1 (GOT1)
α-glutathione S-transferase (GSTα) α-glutathione S-transferase (GSTα)
Sorbitol Dehydrogenase (SDH) Sorbitol Dehydrogenase (SDH)
5′-Nucleotidase/CD73 (5’-NT) 5′-Nucleotidase (5′-NT/CD73).

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Cellomatics Biosciences Ltd. is a specialised Contract Research Organisation (CRO) providing bespoke preclinical laboratory based services.

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