2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 38
Chapter 4: Interferon Bioassays
Cytopathic Effect Assay 101
Interferons are pleotropic cytokines with antiviral, antiproliferative and immunomodulatory activities. Activity
can be assessed by methods that quantify any of these characteristics. For many years, however, the standard
assay has been the protection of cells from the cytopathic effect (CPE) of certain viruses, since this was the
initial activity which defined these molecules. The other reason that the CPE assay is used to characterize the
activity of interferons (IFN) is that it is amongst the most sensitive activity assay, with often >100 fold more
sensitivity than the alternatives. In fact, given that the K
d
of IFN on human cells is ∼0.5 nM and in some antiviral
assays the IFN is effective at 0.5 pM, very little receptor occupancy is required for protection of the test cells
from viral challenge.
virus that kills very close to 100% of the untreated cells in the desired incubation
Many combinations of cells and viruses can be used, but certain pairs tend to time.
be used most frequently. To measure mouse interferons, researchers usually The plates are monitored microscopically occasionally during the incubation
use L929 cells with either Vesicular Stomatitis Virus (VSV), often the New time. Once viral killing is complete, the assay is developed. The classic, and still
Jersey strain, or Encephalomyocarditis Virus (EMCV). This cell line is sensitive most common, method involves fixing and staining the plates with an
to alpha, beta and gamma interferons. For the measurement of human acid/methanol solution containing Crystal Violet dye. This dye will stain any
interferon, a wider variety of systems are used. Many researchers will use the remaining cells attached to the plate. Alternatively, vital stains such as neutral
human lung carcinoma cell line A549 due to their ease of growth and relatively red can be used. Assays can also be developed using dyes such as XTT, MTT or
high sensitivity to all forms of human interferons. These cells are usually MTS which measure mitochondrial activity or surviving cells. In the crystal
challenged with EMCV. Human alpha interferon can be assayed using the violet assay, the excess stain is washed off the cells with water or PBS, and the
bovine kidney line MDBK challenged with VSV. One advantage of this system is plate is then dried.
the relative insensitivity of the bovine cells to human IFN-β and IFN-γ giving
some selectivity in uncharacterized samples. Human IFN-β can also be
measured on the green monkey kidney cell line Vero challenged with VSV. The
advantage of this assay is that human IFN-αis much less active on these cells
than IFN-β.
Treatment of cells with IFN leads to the production of a host of proteins,
many of which interfere with viral replication. As noted above, very little
receptor occupancy is required to lead to effective protection, but some time is
required for the synthesis of the new proteins. Generally, IFN is pre-incubated
with the cells for four (rapid assay) to 24 hours (sensitive assay) prior to viral
challenge. Depending upon the cell and virus combination, the cells are then
incubated for a further 24-48 hours to effect nearly complete killing of the
unprotected cells.
CPE assays are generally developed as follows. Cells are plated in 96 well
tissue culture plates at defined density. This density is chosen to ensure that
cells not challenged with virus are just confluent at the end of the assay.
Interferon is titrated by two fold dilution across the plate (12 data points) in Figure 1: A549/EMCV CPE Assay for IFN Alpha. Rows B and C contain an
duplicate or triplicate. Often, a pre-dilution is required. The goal is to dilute the unknown sample. Row F contains the IFN lab standard. Row G, well 1-6 are the
sample sufficiently to have the interferon concentration range from protecting cell control and wells 7-12 are the virus control.
the cells to not protecting near the center wells of the plate. In addition, a
standard is included to allow determination of the activity in Units/ml. These Once dry, the plate is examined microscopically. Wells are examined and the
standards are generally laboratory standards which have been calibrated by well in which 50% of the cells are alive, the endpoint, is noted. This is also noted
numerous repeated assays using one of theinternational standards. Also for the standard. Since the unitage of the standard is known, it can be used to
included is one row which receives no IFN. After incubation of the cells with IFN, calculate the U/ml of the unknown sample. For example, a standard of 2500
virus is added to all the wells, which received either IFN sample or standard. Virus U/ml reaches an endpoint at well 4 on the plate. If the unknown sample has not
is also added to six wells in the row without IFN, while the other six wells receive been pre-diluted and reaches and endpoint at well 9, it contains 32 times the
media with no virus. The six wells receiving virus but no IFN serve as the “virus protective activity based on fold dilution/well raised to the number of
control” and provide the control for 0% protection. The six wells receiving no IFN intermediate wells between the two (i.e., 2
5
= 32). 2500 U/ml standard
and no virus serve as the “cell control” and provide the control for 100%
endpoint times 32 yields 80,000 U/ml for the concentration of the interferon in
protection. The amount of virus to be added is determined empirically. Usually,
the unknown. Sometimes, a sample will reach an endpoint between wells. For
the virus will be added between 0.1 and 1 MOI and approximates the amount of
example, if there is 80% protection in well 9 and 20% protection in well 10, the
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