7 types of bioassays

Bioassays are used to determine the potency of a biopharmaceutical by comparing the biological response related to its mode of action with that of a control preparation. The main considerations for bioassay design will be ease of use, reproducibility and primarily, cost: both financially and in personnel. Primary cells, frozen cells or cell extracts can all be utilized to give robust assay outputs and the days where radioisotopes were the only means of signal detection are, not quite long gone, but could feasibly be phased out in the next decade by all but the most ardent supporters.
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.

Produce and Collecting Ascitic Fluid from Mice

Ascitic fluid (also called ascites) is an intraperitoneal fluid extracted from mice that have developed a peritoneal tumor. For antibody production, the tumor is induced by injecting hybridoma cells into the peritoneum, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities and continue to secrete the antibody of interest, thus creating a high-titered solution of antibodies for collection. Antibody concentrations will typically be between 1 and 10mg/ml.

1. Prime adult female mice (at least 6 weeks old) of the same genetic background as your hybridomas by injecting 0.5mL of pristane (2,6,10,14-tetramethyldecanoic acid) into the peritoneum. These solutions will act as irritants to the mice, which respond by secreting nutrients and recruiting monocytes and lymphoid cells into the area. This creates a good environment for the growth of the hybridoma cells.
2. After 7-14 days, inject 5x10(5) to 5x10(6) hybridoma cells ip. Prior to injection, the cells should be growing rapidly. Centrifuge the cells and wash once in PBS. Inject the cells in no more than 0.5mL of PBS.
3. Ascitic fluid may begin to build up within 1-2 weeks following the injection of the cells. Tap the fluid when the mouse is noticeably large, but before the mouse has difficulty moving. Carefully withdraw as much fluid as possible with 18-gauge needle attached to a 5mL syringe.
4. Return the mouse to its cage. Many mice will produce a second or third batch of ascitic fluid. You can also bleed out the mouse and combine the blood with the ascitic fluid.
5. Incubate the fluid at 37°C  for 1 hour. Transfer to 4°C overnight.
6. Spin the fluid at 3000g for 10min. If there is an oil layer, remove this first and discard. Carefully remove the supernatant from the cell pellet. Spin again if necessary.

A single mouse may yield as much as 10mL of ascitic fluid per batch. Antibody concentrations may be as high as 10mg/mL.

Prepare 0.8 % methyl-cellulose

Prepare 0.8 % methyl-cellulose medium as follows;

1. Add 8 g of methyl-cellulose powder into 500 ml water followed by autoclave to dissolve powder.
2. Prepare 500 ml of 2× MEM media by dissolving MEM powder in water followed by supplement with 2 % heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin and streptomycin and 25 mM HEPES.
3. After filtration of 2× MEM media by 0.2 mm membrane, mix well with 500 ml of prepared methyl-cellulose and store at 4 °C.

Western Blot - Cell Lysate Protocol

This protocol is intended to provide a set of initial conditions for analysis of cell lysates samples by Western blot. Further optimization may be required for individual samples or analytes. Follow manufacturer's protocols for specific reagents when applicable.

Preparation of Cell Lysates for Western blots:

Prepare total cell lysates by solubilizing cells in an appropriate sample buffer, such as 2X SDS sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue), at approximately 2x10⁶-1x10⁷ cells per mL. The extracts are heated in a boiling water bath for 5 minutes and then sonicated with 3-4 bursts of 5-10 seconds each.

Primary Antibody Selection & Optimization

The most important factor when designing an IHC/ICC experiment is selection of the primary antibody. In turn, the critical feature of a primary antibody is specificity for the epitope. All steps of an IHC/ICC experiment must be optimized to visualize specific staining and minimize non-specific background signals. This includes performing initial studies to determine the appropriate incubation conditions for each primary antibody. The working dilution for an antigen affinity-purified polyclonal antibody is generally lower than that of a monoclonal antibody but these values must be determined empirically. To achieve a robust and specific signal, a high quality antibody that exhibits minimal cross-reactivity should be employed.

Starting conditions for Primary Antibody Incubation

	Monoclonal Antibody	Polyclonal Antibody
Tissue	5-25 µg/mL, overnight at 4 °C	1.7-15 µg/mL, overnight at 4 °C
Cells	5-25 µg/mL, 1 hour at room temperature	1.7-15 µg/mL, 1 hour at room temperature
Advantage	Single epitope specificity	Lower concentration required
Limitation	Vulnerable to epitope masking	Heterogeneous population

Protocol for Making a 4% Formaldehyde

The vast majority of IHC/ICC procedures employ fixation of tissues and cells using formaldehyde-based fixatives. The protocol below describes the technique for generating a 4% formaldehyde solution in PBS. The most effective fixative must be determined experimentally.
Caution: Formaldehyde is toxic. Please read the MSDS before working with this chemical. Gloves and safety glasses should be worn and solutions made inside a fume hood.

Please read the protocol in its entirety before starting.

Protocol for the Preparation & Fixation of Cells on Coverslips

Source : www.rndsystems.com