Measuring Interferon Alpha Level

Measuring Interferon Alpha Level

Recombinant Human IFN Alpha Interferon alpha (IFN-α) was one of the first FDA approved biotherapeutic treatments. Since its approval, it was shown to be a powerful cytokine with potent therapeutic activity but unfortunately strong side effects. The overall use of IFN-α increased dramatically when it was approved as the treatment of choice for hepatitis C infection. However, nearly half of the individuals infected with genotype 1 of the virus still fail to respond to therapy.(1) Consequently, several pharmaceutical companies are now trying to turn on the body's own interferon alpha family of proteins using immunomodulatory molecules in hopes this will elicit a more complete antiviral response. Both therapeutic approaches are not without risk due to the side effects associated with IFN-α. Additionally, there is a growing amount of published scientific articles suggesting IFN-α may be involved in certain autoimmune diseases including systemic lupus erythematosus (SLE).(2) Combined, these observations clearly suggest an increased need to monitor IFN-α levels in normal and diseased individuals along with patients undergoing therapy. How much IFN-α produced in response to new immunomodulatory therapies by the body will be functionally equivalent to the current IFN-α exogenous treatment regimen? How much “basal” IFN-α is beneficial to prevent or limit viral infection, and how much is too much, thereby predisposing an individual to autoimmune disorders? Although IFN-α has been studied for over 50 years, we are only now beginning to understand that interferon can have vastly dichotomous activities. This review will highlight the primary methods currently employed to study interferon levels in research and in clinical settings of this very useful, yet only moderately understood cytokine. More extensive detailed procedures may be found elsewhere.(3-6)

 

Measuring interferon-alpha levels.

IFN-α protein levels can be determined by direct methods including directly determining mass levels and indirectly by many unique biological activity assays. There have been many assays developed over the years for the detection of IFN-α. While many were important for increasing our understanding of IFN-α expression and activity, many were very laborious and low-throughput. The assays highlighted here are those of a medium to high throughput in nature that could be adapted to large scale drug screening and/or clinical sample analysis. Both purified IFN-α preparations as well as IFN-α from stimulated cells can be analyzed by the methods described below.

 

Direct protein analysis

The sandwich Enzyme Linked ImmunoSorbent Assay (ELISA) has become an invaluable tool for the rapid and highly quantitative analysis of cytokines including IFN-α. Most of these commercial assays work in complex sample matrices including tissue culture medium, serum, plasma, cerebrospinal fluid and urine. Advantages of this approach are that these assays are rather simple, rapid and they are highly specific for IFN-α. Limitations are the possibility of false positive results due to the presence of heterophilic binding agents and false negatives due to the interaction of the IFN-α with soluble receptors, antibodies or other factors that would inhibit IFN-α capture and detection. One area that is difficult to control is individual IFN-α subtype detection capacity. Many IFN-α ELISA kits are developed to detect IFN-α2. One must make sure when using an IFN-α ELISA kit that it has been developed to detect most if not all of the IFN-α subtypes. This way, you can have accurate global expression data pertaining to the IFN-α family of proteins and just not IFN-α2.

 

An alternative to solid-phase ELISA are the solution based bead-based technology systems where the assay sample is mixed with the anti-IFN-α beads and the detection antibody in solution. The sample is then passed through a reader that detects and quantifies the level of bound IFN. Similar to the more complex ELISA systems, these reagents are more costly and require dedicated reader systems. A distinct advantage of these systems over standard ELISA is the potential to detect multiple cytokines simultaneously within a single sample. Another method which is limited to cells in culture involves the detection of cells expressing IFN-α by flow cytometry methods. This is not as sensitive as ELISA and does require a flow cytometer and a skilled operator. However, these assays are useful when performing studies to look for distinct IFN-α expressing cell populations in mixtures.

 

In summary, the single analyte colorimetric anti-IFN-α continues to be the simplest and most affordable means to monitor IFN-α protein levels. The more expensive and laborious procedures are valid alternatives if the proposals warrant the sensitivity or multiplex capabilities of these approaches.

 

Biological Activity Assays

The assay of IFN-α biological activity by most methods is prone to variation. Early studies conducted during the clinical development of IFN-α2 showed that significant variation was common between different laboratories and operators(7,8). Different cell line origins, passage numbers, media constituents and virus preparations were all sources of possible assay variation. Consequently, reference standards were prepared to be analyzed in parallel with test samples to calculate activity levels. These reference standards are available from several sources and are the utmost critical reagent for any assay method chosen. Since these standards are in finite levels, they are only used to calibrate internal lab standards that are run in all assays each and every time and preferably, on each assay plate.

 

The cytopathic protective effects (CPE) assay continues to be the most widely used assay to determine IFN-α biological activity. Briefly, IFN-α responsive cells are treated with serial dilutions of test samples and know dilutions of lab standards. Cells are then challenged with a single concentration of cytopathic virus. A second incubation step is carried out and completed when cells treated with only virus show complete killing and lysis. IFN-α activity values of the experimental samples are determined by comparing dilution endpoints to those of the lab standards. Advantages of this approach are low reagent costs and high sensitivity. Disadvantages include cross-reactivity with other type I cytokines (and potentially other cytokines depending on the cell line and virus used), extensive training required to perform assays on a reproducible level, multiple addition steps and overall assay times (usually 24 to 72 hrs). Moreover, biological activity can vary greatly in a cell and virus type manner. Therefore, if these experiments are going to be used as screening assays, one must have sufficient stocks of cells and virus to maintain assay continuity over time.

 

Another clinical application of IFN-α proteins is as an anti-cancer agent. Consequently, IFN-α is also screened for the ability to inhibit the replication of cancer cells in culture and therefore a desired screening assay can be used to measure the antiproliferation capacity of IFN-α. These assays are similar to antiviral assays in that they are carried out by treating cells with experimental sample and lab standard dilutions in parallel and activity is determined by comparison of experimental and lab standard endpoints. The advantages of these assays are they require less reagent addition steps than antiviral assays and have good sensitivity. Disadvantages include an extended 3 to 7 day overall assay time and similar to antiviral assays, the potential for cross-reactivity with other cytokines. Nevertheless, these assays are the standard methods for monitoring the antiproliferative effects of interferon-alpha. Another cell-based assay option that indirectly measures IFN-α levels involves monitoring specific gene expression induced by IFN-α in a dose-dependent manner. Preferably, cell lines containing a stably integrated gene construct that contain a promoter region from an interferon stimulated gene (ISG) controlling translation of a reporter gene are employed. Addition of serial dilutions of experimental and lab standards are conducted in parallel and unknown values are determined by backfitting values against the lab standard curve (similar to ELISA data analysis). The primary advantages of these types of assays are comparable sensitivity to antiviral assays without the multiple reagent addition steps and extended incubations times. These assays are simple and rapid when compared to other cell based assays, while still maintaining the sensitivity. Another advantage is that one can employ the use of cryopreserved cells to limit the need for continuous cell culture which also limits assay variability. Disadvantages are similar to all other bioassays in that interfering substances can artificially dampen or enhance the reporter signal. Nevertheless, these newer assays represent a quicker, simpler and more reproducible means to assess IFN-α biological activity. Lastly, flow cytometry can also be employed for indirect IFN-α gene upregulation. Class I major histocompatibility complex is up-regulated by IFN-α. While more labor intensive and less quantitative, it does allow for differentiating the cell types that respond to IFN-α by sorting populations for other cell markers. Therefore this would be a highly useful research tool to determine if IFN-α expression levels could be traced to specific cell types.

 

In summation biological assays, although generally more labor intensive than direct protein assays are critical for the proper evaluation of IFN-α biological activity. One cannot assume biological activity based on protein levels. As such, if activity levels are required for your studies, a bioassay must be carried out. Lastly, the appropriate cell line and additional reagents described for the assays aforementioned need to be carefully evaluated for sensitivity, specificity and reproducibility in relation to the origin of your samples and the assay sensitivities required.

 

Other Options

There are other assays that can be used as well. For example, signal transducer and activator of transcription proteins 1 and 2 (STAT1 and STAT2) are phosphorylated in response to IFN-α treatment and form the core of the transcription unit (along with interferon regulatory factor 9; IRF9) that upregulates ISG expression. These assays can be performed either by ELISA with cell lysates or by high content screening assays. The primary advantages of analyzing STAT1 or STAT2 is that phosphorylation occurs in minutes and assays can be performed within a single day. Drawbacks include the need for cell lysis in the ELISA, and the cost of high content screening equipment. However, the rapidity of these assays makes them a plausible choice especially for screening large compound libraries. In addition, qRT-PCR based detection is possible, but not established for detecting all the IFN-α subtypes. If developed this could be a useful tool for the early and rapid IFN-α production in stimulated cells.

 

Summary

Are there any perfect IFN-α assays? Clearly, there is no single, absolute assay for measuring IFN-α. This is due in part to the nature that they are a family of proteins and also that they are usually expressed with several other pro-inflammatory cytokines that may also produce overlapping biological effects. Perhaps the best approach is to screen both protein mass and biological activity in parallel. Following a series of initial spike recovery studies, it should be possible to have a strong correlation between the two assays. This way, each assay will indirectly serve as an assay control for the other.

 

Today, the advancement of immune response modulating drugs into clinical development is demanding better assays to monitor cytokines like IFN-α. In one instance, TLR agonists are being developed to specifically enhance IFN-α expression, while limiting the expression of TNF-α(9). Likewise, newer cancer therapies are being developed that IFN-α is a critical component. Separately, there is a growing body of evidence that IFN-α may be a causative agent either predisposing or advancing autoimmunity diseases like SLE and that monitoring IFN-α levels may be important for controlling disease progression. A recent search of the NIH clinical trials site www.clinicaltrials.gov using “interferon alpha” as the query returned over 560 hits including recently completed, recruiting and ongoing clinical trials. Monitoring this cytokine will remain an important goal for many areas of therapies for years to come.

 

Lastly, one higher order question remains, are there really “good” and “bad” IFN-α subtype proteins? Would some IFN-α subtypes be better therapies for antiviral and cancer treatments, producing more potent desired activities with fewer side effects? Conversely, could bad IFN-α subtypes associated with autoimmune diseases be selectively blocked, preventing the disease effects but still allowing ample host response to pathogens? These questions cannot be adequately addressed in large scale studies to date because no reagents are commercially available to differentiate all of the individual IFN-α proteins. Hopefully, current studies will help to better determine global levels of IFN-α in a variety of settings so that this information can be used as a base for deconvolution of the individual IFN-α subtypes when these assays are established.

 

References:

  1. Heathcote EJ, “Antiviral therapy: Chronic Hepatitis C”, Viral Hepatitis, Suppl. 1:82-88, 2007.
  2. Pascual V, Farkas L, and Banchereau, “Systemic lupus erythematosus: all roads lead to type I interferons”, Current Opinion in Immunology,18:676-682.
  3. Pestka S, Ed., Interferons, Methods in Enzymology, 78: Part A, 1981.
  4. Pestka S, Ed., Interferons, Methods in Enzymology, 79: Part B, 1981.
  5. Pestka S, Ed., Interferons, Methods in Enzymology, 119: Part C, 1986.
  6. Meager A, “Biological assays for interferons”, Journal of Immunological Methods, 261:21-36.
  7. Pestka S, Meager A, “Interferon standardization and designations”, Journal of Interferon Cytoine Research, 17: Suppl 1:S9-14
  8. Meager A, Gaines Das R, Zoon K, Mire-Sluis A, “Establishment of new and replacement World Health Organization International Biological Standards for human interferon alpha and omega”. J. Immunological Methods. 257 (1-2):17-33, 2001
  9. Thomas A, Laxton C, Rodman J, Myangar N, Horscroft N, Parkinson T, “Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection”, Antimicrobial Agents and Chemotherapy, 51(8):2969-78, 2007.

Find your product of interest by searching for category, species, molecule and/or application.

Visit our blog to read about recent developments in the fields of interferon, cytokine and biomarker research!

Sign up for our mailing list to be kept up-to-date on the latest promotions, company and product news.

Go to top