2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:50 PM Page 22
Application Note
Use of iLite
TM
Human Interferon Alpha Cell-Based Assay Kit and an anti-IFNAR2
neutralizing antibody in rapid activity determination of interferons secreted from
poly I:C-stimulated A549 cells
Xiao-Hong Lin, Dolly Koltchev, Ph.D. and Ronald G. Jubin, Ph.D.
PBL InterferonSource, 131 Ethel Road West, Suite 6, Piscataway, NJ 08854
Background
In recent years, it has become clear that the mammalian innate immune expression, respectively. Type I IFNs seen in stimulated cells include
system responds to numerous pathogen-associated molecular patterns alpha, beta, kappa, and omega, all of which can inhibit viral replication
(PAMPs) produced by many infectious agents. Namely, retinoic acid or induce type I IFN-mediated reporter gene expression. A researcher
inducible gene 1-like receptors (RLRs) and Toll-like receptors (TLRs) has many options to determine the presence (or absence) of type I
have emerged as powerful components that recognize and initiate the IFNs present in the treated cell supernatant. These might include
host immune response to limit pathogen infection and dissemination. quantitative RNA detection assays, classical ELISA kits or using
Among these, nucleic acids have been shown to be highly neutralizing antibodies directed against each individual type I IFN.
immunostimulatory, promoting inflammatory cytokine production However, each of these methods has limitations. For example, several
including interferons. Specifically, double-stranded RNA (dsRNA) is studies have shown that mRNA levels do not always correlate
recognized by several proteins including protein kinase R, 2’5’ effectively with IFN protein levels. Individual ELISA assays and
–oligoadenylate synthetase retinoic acid inducible gene 1 (RIG-1) and neutralizing antibodies to each type I IFN proteins work well, but are
Toll-like receptor 3 (TLR3). Many viruses produce factors to block this laborious and may not be necessary if type IFNs were not produced
host dsRNA response. Consequently, interest in this area of research during the cell treatment. Likewise, performing all of the multiple
has increased dramatically. Therefore, improved methods are needed to assays can use up precious amounts of limited supernatants or serum
more rapidly determine the effects of PAMPs on host control of infection. samples.
One of the classic methods for determining interferon activity units The study presented here is designed to use small quantities of
per ml (U/ml) in treated cells has been the cytopathic effect assay sample in a rapid assay designed to determine if type I IFNs are
(CPE). In this method, cell culture supernatants are collected from present in an experimental sample. Instead of using neutralizing
treated cells and added to naïve cells. Following and incubation period, antibodies to each type I IFN, a single neutralizing antibody that
cells are then challenged with virus. The virus will infect control cells blocks receptor binding of all type I IFNs was utilized to provide a
resulting in cell lysis. If interferon is present, the cells are protected rapid yes/no answer regarding the presence of type I IFNs in a treated
from lysis in a dose-dependent manner. When run in parallel with and cell line.
compared to international interferon standards, the assay effectively
determine the unit level of interferons present in the cell culture Materials and Methods
supernatant (U/ml). The drawbacks of this approach are the need for TLR3 Stimulation
continuous cell culture, highly skilled technicians, and maintaining Human A549 cells (human lung carcinoma) were transfected with lipid
virus stocks. In addition, these assays often take >72hrs to complete. complexes containing increasing concentration of the synthetic dsRNA
Recently, PBL has introduced the iLite
TM
human IFN-α cell-based analogue polyinosinic:cytidilic acid (poly I:C). Six hours after addition of
assay kit (PBL product #51100-1). Similar to the CPE assay, the iLite
TM
the transfection mix, cells were washed, and complete growth media
kit provides the researcher with a U/ml determination of interferon in a (DMEM with 10% FCS) was added and cells were incubated an additional
tissue culture or serum sample. In contrast, this assay does not require 16hrs. Cell culture supernatants were collected for use in the iLite
TM
continuous cell culture, is simple to perform, and does not require the Human Interferon Alpha Cell-Based Assay (PBL product #51100-1).
handling of biohazardous virus stocks. Importantly, it can be
completed in <24hrs. iLite
TM
Assay
This present study was performed to demonstrate the utility of the The iLite
TM
assay was performed according to the kit protocol. Cell
iLite
TM
kit when studying PAMPs, such as dsRNA. It also includes specific culture supernatants were split into three groups. Group 1
conditions for a receptor-neutralizing antibody that researchers in this supernatants received no additional treatment. Group 2 supernatants
and similar areas should find useful when designing their studies. were mixed Mouse anti-Human IFN-α/β R2 receptor antibody (PBL
In either the CPE or iLite
TM
assay format, it is important to determine product #21385-1). Group 3 supernatants were mixed with mouse anti-
the contribution of type I IFNs to the antiviral or the reporter gene human Anti-IFN-γ R1 antibody (PBL product #21580-1).
PBLInterferonSource Toll Free: 1 877- PBL-8881
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