The ProQuantum Human IL-1β Immunoassay Kit is designed to provide highly sensitive quantitative measurements of human IL-1β protein in small sample volumes. Utilizing proximity ligation assay (PLA) technology, the assay combines the analyte specificity of high-affinity antibody-antigen binding with the signal detection and amplification capabilities of real-time PCR to achieve a simple yet powerful next-generation protein quantitation platform. A user-friendly workflow combined with intuitive Cloud-based software for analytics enables sample-to-answer in just 2 hours.
• High sensitivity—detect low levels of protein with greater sensitivity than traditional methods
• Broad dynamic range—≥5 logarithmic units, minimizing sample dilutions to ensure they fall within the range
• Small sample consumption—use 2–5 µL of sample (compared to 75 µL for triplicate wells with other methods)
• Fast, easy workflow—2 hours from sample to answer, with no wash steps
• No proprietary instrument to purchase—runs on any real-time PCR instrument
ProQuantum immunoassays utilize a matched pair of target-specific antibodies, each conjugated to a DNA oligonucleotide. During antibody-analyte binding, the two DNA oligos are brought into close proximity, which allows for ligation of the two strands and subsequent creation of a template strand for amplification. This platform leverages the sensitivity and large dynamic range of Applied Biosystems TaqMan real-time PCR technology (Figure 1).
The assay workflow is fast and easy—2 steps in 2 hours. There are a total of 7 components in each kit (Figure 2). First, mix the antibody-conjugates, dilute the sample, and create the standard curve in a working plate. Then, using a multi-channel pipette, add the antibody-conjugates and sample (or standard) into the wells of a PCR plate and incubate for 1 hour. Combine the master mix and ligase and add to the wells of the PCR plate, then run the plate on any qPCR instrument. After the run is complete, import the results file into the ProQuantum cloud-based software at https://apps.thermofisher.com/apps/proquantum. Using this software, the data can be analyzed easily to obtain protein concentration values. The software allows you to set up standard curves, design plate layouts, set up customized assay instructions, and obtain robust statistical group-wise comparisons.
Interleukin-1 beta (IL-1 beta) is a proinflammatory cytokine expressed by monocytes, macrophages, and dendritic cells. IL-1 beta is synthesized in response to inflammatory stimuli as a 31 kDa inactive pro-form that accumulates in the cytosol. Cleavage of pro-IL-1 beta into the active 17 kDa protein requires the activation of inflammasomes, which are multi-protein complexes that respond to pathogens, stress conditions, and other danger signals. Inflammasome activation triggers the processing of the caspase-1 precursor into its active form, which in turn cleaves pro-IL-1 beta. IL-1 beta lacks a signal sequence peptide for classical ER/Golgi pathway and is secreted alongside caspase-1 via an alternate and incompletely understood mechanism. Although IL-1 beta is most often secreted in its active form, secretion of the uncleaved protein may be detectable under some biological conditions. IL-1 beta signals through two receptors, IL-1RI and IL-1RII, both of which are shared with IL-1 alpha. IL-1 beta activity can be moderated by IL-1 Receptor Antagonist (IL-1RA), a protein produced by many cell types that blocks receptor binding through competitive inhibition. IL-1 beta play an important role in innate host defense by triggering the production of other proinflammatory cytokines in target cells and initiating acute-phase responses to infection and injury. Elevated levels of IL-1 beta have been associated with many chronic inflammatory conditions IL-1 beta neutralizing antibodies potential therapeutic value.
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