2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:50 PM Page 31
Application Note
Use of a polyclonal antibody to neutralize the activity of human interferon alpha on cell
lines of different species origins.
Jessica Esposito, Thomas Lavoie, Ph.D. and Ronald Jubin, Ph.D.
PBL InterferonSource, 131 Ethel Road West, Suite 6, Piscataway, NJ 08854
Background
Interferons are potent cytokines that can inhibit viral replication, limit cell grown in MEM supplemented with 10% FCS as were the MDBK (bovine kidney
proliferation and enhance activation of the adaptive immune responses. Most cells). Cells were plated at subconfluent levels the day before neutralization
interferons display strong species specificity. However, closer species experiments were initiated.
relationships tend to lead to higher cross-reactivity. For example, non-human Neutralization Assay: Human IFN-α2b at 50 U/ml (PBL product #11105-1) was
primates respond to human IFNs both in vitro and in vivo. In contrast, human IFN- combined with dilutions of the neutralizing antibody rabbit anti-human IFN-αfor
α2 has an EC
50
of <1 U/ml on human A549 cells, while the EC
50
on murine cells 60 minutes then the IFN-α/PAb mixture was added to each respective cell line.
is >1000 U/ml. One exception to this phenomenon is the discovery that Madin- The cells were subsequently returned to the incubator for 20 hrs. Each cell line
Darby bovine kidney cells (MDBK) are broadly cross-reactive to human IFN-α was then subjected to viral challenge as follows: A549 cells were infected with a
including human IFN-α2. Consequently, researchers who work on human 1:10,000 dilution of Encephalomyocarditis Virus (EMCV) and MDBK cells were
interferons can find themselves working on multiple cell lines of different species infected with a 1:100,000 dilution of Vesicular Stomatitis Virus (VSV), while the
lineages. One useful technique used to prove the biological activity of human IFN- LLC-MK2 cells were challenged with a 1:10,000 dilution. Plates were incubated for
αis to conduct a sample analysis with and without an antibody that neutralizes 24-36 hours following addition of virus cells were stained with crystal violet. The
IFN-α. One caveat is the substantial sequence variation between the type I IFN cell density was determined by OD
450
measurement on a Molecular Devices Vmax
receptors expressed on human, non-human primate and especially bovine cells. plate reader. Neutralization titers were determined by the following formula:
Therefore, one cannot assume that the IFN:receptor interactions behave the same Divide IFN-αunits/well by serum dilution/well.
on each cell line. This study was conducted to determine if a rabbit polyclonal
antibody raised against human IFN-α2 would inhibit the activity of human IFN-α2 Results
on human (A549), NHP (LLC-MK2) and bovine (MDBK) cells. The addition of IFN-α2 at 50 U/ml resulted in complete protection of each cell line
tested when challenged with the appropriate quantity of virus. In contrast, the cells
Materials and Methods which did not receive IFN-α2 displayed complete loss of monolayer. The addition of
Cell Lines: Human A549 cells (human lung carcinoma) were maintained in F12 polyclonal antibody directed against human IFN-α2 displayed similar dose-
media supplemented with 10% FCS. LLC-MK2 (rhesus monkey kidney cells) were dependent loss of antiviral activity regardless of cell type or virus.
Sample A549 Neutralizing Titer MDBK Neutralizing Titer MK2 Neutralizing Titer
Rabbit anti-human IFN-α 3.1 x 10
5
U/ml 4.3 x 10
5
U/ml 1.3 x 10
5
U/ml
Anti-Human Alpha Titration Protection by Alpha IFN
n
i
o
n
i
o
e
ct
e
ct
P
r
ot
P
r
ot
% %
PAb Diluton [IFN] log (U/ml)
Discussion
Human IFN-αstudies are primarily carried out on human cells. However, there are demonstrate that the effects are specifically due to interferon. However, it is not
instances when other cell lines are utilized. MDBK cells have proven useful since clear if the IFN-receptor interaction between each cell line tested could be
they respond to human alpha interferons, but not to beta interferon. In contrast, inhibited by a polyclonal antibody. This study clearly indicated that the polyclonal
non-human primate cells are often used as a prelude for more formal in vivo antibody raised in rabbits against human IFN-α2 could indeed interact with the
studies in monkeys. Neutralizing antibodies provide a useful tool to protein to sufficiently inhibit protein activity on all cell lines tested.
www.interferonsource.com Tel: +1 732-777-9123
I
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