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Immunoassays | Inflammation & Cytokines

Erenna® IL-8/CXCL8 Immunoassay Kit (Cat# 03-0055-xx)

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Introduction

The Erenna® IL-8/CXCL8 Immunoassay uses a quantitative fluorescent sandwich immunoassay technique to measure IL-8 in human plasma samples. A capture antibody specific for human IL-8 has been pre-coated onto paramagnetic microparticles (MPs). The user pipettes MPs, standards, and samples into uncoated microplate wells. During incubation, the IL-8 present in the sample binds to the capture antibody on the coated MPs. Unbound molecules are washed away during the subsequent buffer exchange and wash steps. Fluor-labeled detection antibody is added to each well and incubated. This detection antibody recognizes and binds to IL-8 that has been captured onto the MPs. During the following wash step the MPs are transferred to a clean plate. Elution buffer is then added and incubated. The elution buffer dissociates the bound protein sandwich from the MP surface, releasing the labeled antibodies. These antibodies are separated during transfer to a final microplate. The plate is loaded into the Erenna® System where the labeled molecules are detected and counted. The number of fluor-labeled detection antibodies counted is directly proportional to the amount of IL-8 present in the sample when captured. The amount of IL-8 in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® IL-8/CXCL8 Immunoassay Kit1
Lower Limit of Detection 0.07 pg/mL
Lower Limit of Quantification2 0.49 pg/mL
Upper Limit of Quantification 250 pg/mL
Low-end CV% Range 6 - 8%
Low-end CV% Average 12%
Recommended Sample Volume 50 μL
Minimum Sample Volume Required3 10 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery
3 based upon median [IL-8] in a healthy reference


FIGURE 1. [IL-8] in EDTA plasma from 10 healthy donors, with median and interquartile range. The Erenna® IL-8 Immunoassay Kit reliably quantifies IL-8 in healthy subjects, who have a median [IL-8] of 3.6 pg/mL that is well above the detection limit of 0.07 pg/mL.

FIGURE 2. Erenna® IL-8 Immunoassay Evaluation Reagent Kit low-end standard curve signal (left) and curve fit (right).

Representative data shown for demonstration purposes only. Individual results may vary depending upon samples tested and protocol used.

Biology and Disease

Interleukin-8 (IL-8) is a CXC chemokine synthesized by many cell types, including macrophages, epithelial cells and endothelial cells in response to a pathogen or inflammatory stimulus. IL-8 functions as a chemotactic factor that attracts neutrophils, basophils, and T-cells. IL-8 is one of the major mediators of the inflammatory response and of innate immunity. Its function is to recruit neutrophils to phagocytose the foreign antigen. IL-8 is also a potent angiogenic factor that induces neutrophils to leave the bloodstream and enter into the surrounding tissue. IL-8 has been shown to have roles in the pathological angiogenesis that is associated with solid tumors, diabetic retinopathy, and rheumatoid arthritis (RA).


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Interleukin-8
UniProtKB/Swiss-Prot ID: P10145
Protein-protein interaction database: P10145
Alternative Names: C-X-C motif chemokine 8, Emoctakin, Granulocyte chemotactic protein 1, Monocyte-derived neutrophil chemotactic factor, Monocyte-derived neutrophil-activating peptide, Neutrophil-activating protein 1, Protein 3-10C, T-cell chemotactic factor
Gene Names: IL8
Synonym Gene Names: CXCL8
Function: IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to an inflammatory stimulus. IL-8(6-77) has a 5-10-fold higher activity on neutrophil activation, IL-8(5-77) has increased activity on neutrophil activation and IL-8(7-77) has a higher affinity to receptors CXCR1 and CXCR2 as compared to IL-8(1-77), respectively.
Biological Processes: G-protein coupled receptor protein signaling pathway, angiogenesis, calcium-mediated signaling, cell cycle arrest, immune response, induction of positive chemotaxis, inflammatory response, negative regulation of cell proliferation, neutrophil activation, neutrophil chemotaxis, regulation of cell adhesion, regulation of retroviral genome replication
Molecular Function: chemokine activity, interleukin-8 receptor binding

* Source Info from www.uniprot.org.


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