BioOutsource

Examples of BioPotency Assays

Cell Death Assays

LS-L929 CYTOTOXICITY ASSAY

This test utilises a simple cell culture-based cytotoxicity (in which cell viability is determined by uptake of a dye). It is based on the premise that cell death is an unequivocal indication of product toxicity. Mouse fibroblasts grown in suspension are exposed to product over a range of concentrations for set periods of time. After exposure to a test compound, viable cells may be identified by their ability to accumulate dye.

Basic Procedure

Mouse fibroblasts are maintained in continuous culture, diluted when required and known volumes incubated in the presence of test material in a range of concentrations for a specified time. The cells are then examined by the addition of dye. An analyser (together with the correct filter systems) enables differentiation between viable and non-viable cells. The endpoint is taken as 50% cell death and this is used to determine the Effective Concentration (EC50) of test substances. The choice of cell type is, to a certain extent is arbitrary. Cell types can be chosen for specific physiological and biochemical properties to form the basis for second-order tests.

Cell Proliferation

TF-1 PROLIFERATION ASSAY

Traditionally, cell proliferation assays have measured the tritiated thymadine incorporation by cells following stimulation with the product of interest. Novel kits are now available that measure the proliferation inducing properties of product that is measured with a secondary indicator such as ATP. This test is based on the ability of the TF-1 cell line (human lymphoblast cell line) to respond to a number of active biologics such as Erythropoeitin and Granulocyte Macrophage Colony Stimulating Factor. Cells grown in suspension are exposed to product over a range of concentrations for set periods of time. After exposure to a test compound, cell concentration is determined relative to ATP concentration.

Basic Procedure

TF-1 cells are maintained in continuous culture, diluted when required and known volumes incubated in the presence of test material in a range of concentrations for a specified time. The cells are then examined by cell solubilisation and the measurement of the concentration of ATP. A luminometer enables the concentration of ATP to be accurately measured. The endpoint is taken as 50% cell proliferation when compared to the appropriate control cell cultures and this is used to rank the Effective Concentration (EC50) of test substances. There are other cell types from different animal species of that can be substituted where appropriate.

Cell Reporter Assays

ENGINEERED JURKAT REPORTER GENE ASSAYS

This test is based on the ability of the reporter cell line (a product specific modified line, typically based on Jurket cells) to specifically respond to the target biologic. The assay measures the response of a modified cell, usually with a Luciferase insert, to the presence of product that is measured with a secondary indicator such as Luciferase or other enzymatic activity. Cells grown in suspension are exposed to product over a range of concentrations for set periods of time. After exposure to a test compound, viable cells may be identified by the response of the reporter gene.

Basic Procedure

Cells are maintained in continuous culture, diluted when required and known volumes incubated in the presence of test material in a range of concentrations for a specified time. An luminometer (together with the correct filter systems) enables the concentration of the reporter gene product to be accurately measured. The endpoint can be taken as 50% reporter gene response when compared to the appropriate control cell cultures and this is used to rank the Effective Concentration (EC50) of test substances. There are many reporter cell types from human or different species of animal that can be substituted where appropriate.

Binding Assays

Binding assays can be divided into two areas that either demonstrate the functional activity of a product or simply the binding of the product to a native or modified cell line or isolated protein. Screening assays usually utilise the simple binding model whereas the functional activity of product invariably has to be demonstrated for release assays. In this instance, the assay would detect the expression of a surface protein in response to the application of the product to the cell line. The examples given here relate to flow cytometry, Biacore and ELISA assays, however other technologies exist including electrochemiluminesence.

FLOW CYTOMETRY BINDING ASSAYS

Flow cytometry is a means of measuring certain physical and chemical characteristics of cells as they travel in suspension one by one past a sensing point. Physical characteristics such as cell size, shape and cell component or function, such as a bound and labelled antibody can be detected.

Basic Procedure

Cells are maintained in continuous culture and are introduced to the test material at a range of concentrations for a specified time. The test material can be labelled directly or a secondary immunoprobe can be used to detect the presence of bound test material. A flow Cytometer enables the concentration of the cells expressing the reporter fluorescent probe to be accurately measured in relation to the unbound cells. The endpoint can be taken as the concentration of product that achieves a specified percentage of cells responding.

The FACS system has the ability to measure a large variety of different aspects of a cell including, but not limited to the following; DNA content, DNA composition, DNA synthesis, cell cycle stage, chromatin structure, RNA, protein, sulphydryl groups, Antigens (surface, cytoplasmic & nuclear), lectin binding sites, cytoskeletal components, membrane structure (potential, permeability & fluidity), enzyme activity, endocytosis, surface charge, receptors, bound and free calcium, apoptosis, necrosis, pH, drug kinetics.

BIACORE or ELISA BINDING ASSAYS

In the ELISA system the test material to be tested is introduced into the system through capture by an antibody specific to the product. Following immobilization of the product the detection antibody is added, forming a complex with the product. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. After the final wash step the plate is developed by adding an enzymatic substrate to produce a detectable signal that is proportional to the quantity of antigen in the sample.