Background

In recent years, it has become clear that the mammalian innate immune system responds to numerous pathogen-associated molecular patterns (PAMPs) produced by many infectious agents. Namely, retinoic acid inducible gene 1-like receptors (RLRs) and Toll-like receptors (TLRs) have emerged as powerful components that recognize and initiate the host immune response to limit pathogen infection and dissemination. Among these, nucleic acids have been shown to be highly immunostimulatory, promoting inflammatory cytokine production including interferons. Specifically, double-stranded RNA (dsRNA) is recognized by several proteins including protein kinase R, 2’5’–oligoadenylate synthetase retinoic acid inducible gene 1 (RIG-1) and Toll-like receptor 3 (TLR3). Many viruses produce factors to block this host dsRNA response. Consequently, interest in this area of research has increased dramatically. Therefore, improved methods are needed to more rapidly determine the effects of PAMPs on host control of infection.

One of the classic methods for determining interferon activity units per ml (U/ml) in treated cells has been the cytopathic effect assay (CPE). In this method, cell culture supernatants are collected from treated cells and added to naïve cells. Following and incubation period, cells are then challenged with virus. The virus will infect control cells resulting in cell lysis. If interferon is present, the cells are protected from lysis in a dose-dependent manner. When run in parallel with and compared to international interferon standards, the assay effectively determine the unit level of interferons present in the cell culture supernatant (U/ml). The drawbacks of this approach are the need for continuous cell culture, highly skilled technicians, and maintaining virus stocks. In addition, these assays often take >72hrs to complete.

Recently, PBL has introduced the iLite™ human IFN-α cell-based assay kit (PBL product #51100-1). Similar to the CPE assay, the iLite™ kit provides the researcher with a U/ml determination of interferon in a tissue culture or serum sample. In contrast, this assay does not require continuous cell culture, is simple to perform, and does not require the handling of biohazardous virus stocks. Importantly, it can be completed in <24hrs.

This present study was performed to demonstrate the utility of the iLite™ kit when studying PAMPs, such as dsRNA. It also includes specific conditions for a receptor-neutralizing antibody that researchers in this and similar areas should find useful when designing their studies.

For research use only, not for diagnostic or therapeutic use.