Measuring the concentration of total interferon-alpha secreted by cells in peripheral blood samples, serum samples or even culture supernatants is not an easy task. There are 14 different isoforms of this powerful stimulatory cytokine that are secreted by different cells in response to different stimuli. It is therefore very difficult to detect all the different isoforms using one single assay and also difficult to know which assay will be best for each cell type following stimulation. The Human Human IFN-á Platinum ELISA kit from eBioscience is the only commercially available ELISA kit that allows you to measure a broad enough spectrum of the most frequently expressed IFN-alpha isoforms, from an array of different sample sources, in order to realistically quantify total IFN-alpha secretion.
This ELISA kit includes enough reagents to analyze 96 samples in a flexible manner. 12 strips of 8 wells are provided that can be clipped into the provided frame and allow for as many or as few samples to be analyzed as are required. This assay is able to detect all type 1 and type 2 IFN-alpha isoforms, recognized to be the most commonly expressed during immune responses and account for more than 60 % of all IFN-alpha protein produced. There is also no cross-reactivity with IFN-beta, IFN-gamma, TNF-alpha or TNF-beta with this assay, allowing the user to obtain more robust data.
The manual is not the most simple to follow and requires the user to flip back and forth from one page to another in order to find relevant information when making the various reagents and wash buffers. Once this is overcome, however, the protocol is relatively simple to follow.
I have found that combining the incubation of the secondary HRP-conjugated antibody with the initial incubation of the sample dramatically reduces the time taken to complete the protocol and the number of wash steps required. Reducing the number of washes allows the user to produce more reliable standard curves (Figure 1) and duplicate results with less variation. As with all ELISA kits, care must be taken to not allow the substrate solution to sit for too long at room temperature or in direct light before use; however, this is the only major consideration that must be taken with the reagents provided.
Figure 1. Example of a standard curve generated using the eBiosciences' Interferon-alpha Platinum ELISA kit using the supplied reagents. The efficiency of this standard curve is 99.8 % and would therefore allow the user to quantify their samples with confidence.
I used this kit to examine whether plasmacytoid dendritic cells isolated from peripheral blood undergo maturation normally, as characterized by their secretion of IFN-alpha, when exposed to various stimuli. Unlike other ELISA kits, this kit allowed me to measure the amount of IFN-alpha present in the peripheral blood that the cells were isolated from (in order to show they were not already activated) and subsequently also allowed me to measure the secretion of a broad range of IFN-alpha isoforms from tissue culture supernatants following stimulation.
In summary, this is an extremely versatile kit that allows the user to complete an assay for the most abundant isoforms of IFN-alpha in one single assay. In addition, this assay can be completed in a far shorter time than other ELISA assays. The reagents supplied are all of a high quality and this allows the kit to produce reliable results that the user will have confidence in.