Assay Services in South Africa

Assay Services

All assays are performed using established methods published in peer-reviewed journals.

Cytotoxicity Assays

Cytotoxicity is defined as the ability of an agent to produce a toxic effect on a cell. Cytotoxicity assays are used to test the ability of cells to continue proliferating in the presence of a test compound or substance over a specific time period.

Cell based viability assessment with the choice of various tumorigenic and non-tumorigenic cell lines. Two end point analysis options are available:

• MTT (metabolic activity)

• Hoechst 33342/Propidium iodide dual staining analysed using the ImageXpress Micro XLS

Genotoxicity Assays

One of the most reliable assays for the identification of potential carcinogens is the micronucleus assay. A micronucleus forms when a fragment of a chromosome or a whole chromosome separates from the rest of the nucleus during mitosis. The following assay is available for genotoxicity screening:

• Micronucleus quantification using the ImageXpress Micro XLS and a fluorescent nuclear dye.

Anti-Proliferation Assays

Various assays are available to explore the molecular mechanism of anti-proliferatory samples. Anti-proliferation analysis will provide information on the mechanism of cell death induced by a sample and suggest anti-cancer potential. Mechanism based assays include:

• Cell cycle analysis

• Apoptosis (caspase activation, annexin V binding, sub-G1 DNA content, mitochondrial depolarisation)

• Senescence (SA-β-GAL activity)

• Autophagy (acidic vacuole quantification)

Numerous cell lines to choose from including: A549, B16F10, MeWo, HT-29, Caco-2. MCF-7, HeLa, PC3, Mia-PaCa, HepG2, U937 and Jurkat.

Anti-Diabetic Assays

Diabetes is a multifactor disease and consequently many potential therapeutic targets exist. Various target directed screening assays addressing the most prominent anti-diabetic mechanisms including, postprandial hyperglycaemia, insulin sensitivity, glucolipotoxicity, adipocyte function and inflammation are available. These specific anti-diabetic assays are available:

• Inhibition of carbohydrate digestion (alpha-amylase and alpha-glucosidase inhibition)

• Glucose utilization (Hepatocyte, adipocyte and myocyte)

• DPPiv inhibition

• Protein glycation inhibition

• Anti-inflammatory activity-(NO production in RAW 264.7 cells)

• β-cell function (proliferation, glucolipitoxicity)

• Adipogenesis (3T3-L1 differentiation)

Anti-Microbial Assays

The search for new anti-microbial agents has increased due to an increase in occurrence of antibiotic resistant pathogenic bacteria, leading to treatment failure. Anti-microbial susceptibility testing can be utilized for drug discovery and the prediction of therapeutic outcome. There are numerous methods for the screening of biological extracts for potential anti-microbial activity.

• The microbroth dilution susceptibility method

• The p-Iodonitrotetrazolium chloride (INT) assay to determine the minimum inhibitory concentration (MIC) of biological extracts using INT dye

Anti-TB Assays

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis and recognised as one of the leading causes of death (De Silva et al., 2013). Due to the increase in multi-drug resistant tuberculosis, there is a need for rapid, high-throughput, cost-effective assays to screen new drug candidates. Various approaches can be employed to evaluate the antibacterial activity of compounds which include micro-broth dilution and agar diffusion methods.

• Microplate Alamar Blue assay (MABA), is a colorimetric assay that is routinely used for antimycobacterial drug screening.

Anti-Oxidant Assays

The various aerobic metabolic processes can generate toxic by-products or free radicals, such as reactive oxygen (ROS) and reactive nitrogen species (RNS). These free radicals are highly reactive relative to their molecular forms. Therefore, under normal conditions, free radical levels can be controlled and detoxified by the anti-oxidants (AO) found in the body. However, the uncontrolled production and presence of free radicals culminates in oxidative stress and cell death.

AO capacity can be used as a parameter for medicinal bioactive constituents, using a range of AO methodologies that assess according to the type of anti-oxidants measured (lipophilic or hydrophilic, enzymatic or non-enzymatic), type of reagent used (radicalic or non-radicalic), solvent character (aqueous or organic) or by mechanism of reaction (hydrogen atom transfer or electron transfer).

The in vitro assays available are:

• DPPH method

• FRAP assay

• ORAC

• CAPe

• NO scavenging

• Cellular oxidative stress protection using CellRox

• Mitochondrial membrane potential protection using TMRE

Drug-Interaction Assays

As a result of their broad substrate specificity, inhibition of drug metabolising enzymes by one drug often adversely affects the metabolism of another drug. In vitro testing can provide important information to prevent herb-drug or drug-drug interactions.

These specific drug interaction assays are available:

• CYP3A4 inhibition

• Carboxylesterase inhibition

• Beta-glucuronidase inhibition

Wound Healing Potential

The skin forms a protective barrier and acts as the primary defence mechanism preventing the invasion of pathogens from the external environment. Disruption to the integrity of the skin initiates a multistep process that ultimately leads to reconstruction of the damaged area and reestablishment of this barrier function. Due to the complexity of the wound healing process, numerous opportunities for therapeutic intervention to improve wound repair exist. Assays used for the determination of wound healing potential are:

• Collagenase inhibition

• Targeting of inflammatory intermediates

• Targeting of fibroblast function

Anti-Inflammatory Activity

Although inflammation is usually associated with a protective or healing response, many chronic diseases are characterised by persistent inflammation ultimately resulting in tissue dysfunction. For this reason, the anti- or pro-inflammatory activity of test samples need to be considered within the context of the disease in question as well as the disease progression stage at which intervention will be considered, in order to accurately evaluate the potential therapeutic significance. Furthermore, multiple mechanisms may collectively contribute to an inflammatory response; consequently it is necessary to take into account that a single target specific in vitro model does not assess the total domain of potential therapeutic activity.

In vitro assays used for the determination of anti- or pro-inflammatory activity are:

• Nitric oxide production

• NF-κB nuclear translocation

• COX-2 and iNOS antibody staining

• Phagocytosis

Hepatotoxicity

Animal testing for hepatotoxicity is associated with many disadvantages, including high cost, ethical considerations as well as low predictive power relating to human hepatotoxicity. In vitro assays allow screening of large numbers of samples at lower cost and without the ethical dilemma associated with in vivo testing. Over the past two decades, improvements in technology and scientific methods have vastly increased the predictive power of in vitro hepatotoxicity methods and pharmaceutical companies are relying on these as a first line of screening to reduce unnecessary animal testing. Cell number and viability are essential parameters to include in an in vitro hepatotoxicity screen as well as mitochondrial dysfunction, steatosis and phospholipidosis. Hepatoxicity assays include:

• Hoechst 33342/Propidium iodide dual staining analysed using the ImageXpress Micro XLS

• Measurement of reactive oxygen species (ROS) using CellRox¬TM

• MitoTrackerTM Green and TMRE staining

• LipidToxTM Red and Green staining

• LysoTrackerTM Red staining

Custom Designed Expriments

We also design experiments according to the needs of the researcher and can combine many assays in one complete series of experiments as required. Please email us for more information.