Here we introduce the 7 main types of cell-based assay which can help you in your quest for that elusive blockbuster.
1. Intracellular signalling
The search for new drugs has led to the need to increase our understanding of the basis of cellular pathways, in order to find new druggable targets. The benefit of targeting intracellular signalling to bioassay design is that inhibiting a signal point in a signalling cascade can affect a wide variety of downstream proteins. These proteins can be measured in cells as active (i.e. phosphorylated) or inactive (native) forms via a number of techniques such as immunofluorescence, ELISA or western blotting, all of which can be scaled up for high throughput.2. Proliferation assays
With proliferation assays, target cells are treated with the biologic drug. The duration is dependent of the mechanism of drug action, its bioavailability and how it is likely to affect the host cells. The final readout for this assay is a cell count – this can be measured using spectroscopic techniques such as fluorescence, luminescence or colorimetric assays.3. Cytoxicity assays
It is advisable to measure two parameters in a cytotoxicity assay - namely live and dead cells after a suitable incubation with your biologic drug candidate. Assay optimization can involve different time frames and this will be dependent on what you expect your drug to do. Live and dead cells can be measured using differential uptake of dyes - live cells tend to actively pump out any xenobiotic dyes, whereas dead cells remain perfect substrates for colorimetric dyes, such as trypan blue or fluorescent DNA intercalating dyes (e.g. propidium iodide).There are two types of cytotoxicity that can be present:
Apoptosis – cells undergo a naturally occurring programmed process of controlled death; controlled biochemical events cause the cell characteristics to change and usually die. Changes might include cell shrinkage, cell fragmentation and global mRNA decay.
Necrosis – cells present in living tissue suffer premature death due to external factors such as cell lysis, infection, toxins, etc. Necrosis is typically very detrimental to the organism and usually results in death.
4. Antibody dependent cell-mediated cytoxicity
Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) is an immune response leading to lysis of target cells, mediated by antibodies specifically binding to surface antigens. It should be noted that not all antibodies have ADCC activity. Several assays are available for determining the efficacy of antibodies or effector cells in eliciting ADCC. These include radioisotope (i.e. chromium-51), fluorescent (europium) release or reporter gene assays. Target cells expressing a certain surface antigen are incubated with the therapeutic antibody specific for that antigen along with, for instance, chromium 51.5. Complement dependent cytoxicity
The complement system is part of the innate immune system, whereby a series of inactive small circulatory blood proteins become activated by antigen/antibody binding. Several assay formats can be utilized for determining the efficacy of antibodies in eliciting CDC. Target cells expressing specific surface antigen are incubated with the therapeutic antibody CDC requires the engagement of the complement cascade via binding to complement C1q, which for the bioassay can be provided using serum or a cocktail of recombinant complement proteins.6. Antibody dependent cell phagocytosis
Phagocytosis is the major immune process for removing pathogens and cell debris. Antibody-Dependent Cell Phagocytosis (ADCP) harnesses this innate immune system to destroy cells which are tagged by a therapeutic antibody. Unlike cytoxicity assays, antibody dependent cell cytoxicity, and complement dependent cytoxicity, antibody dependent cell phagocytosis does not cause direct lysis of the target cell but instead induces monocytes or macrophages to engulf it as part of the mechanism to induce cell death or clearance.7. Migration and colony formation assay
Cell migration is fundamental to many important biological and pathological processes such as wound healing, embryonic tissue reorganisation, chronic inflammation, tumor metastasis and angiogenesis. Cell migration is initiated by stimuli that activate signalling pathway cascades leading to cellular polarization and reorganisation of actin filaments and microtubules. Drugs that effect cell migration have a number of applications and can all be tested by relatively high throughput assay formats.Choosing a robust, reliable fit-for-purpose bioassay in biologics drug development is an essential component in delivering the biologic drug product. It is never too early to move into biassay development as a panel of assays may help you solve the mechanism of action of your drug. A contract research partner experienced in developing bioassays for a large range of different drugs should be able to advise you of what will be needed for your particular situation and can work with you throughout the development of your drug.