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Immunoassays | Prototype

Erenna® IL-7 Immunoassay Prototype Assay (Cat# 03-0054-AA)

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Introduction

The Erenna® IL-7 Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure IL-7 in plasma and serum samples. A capture antibody specific for IL-7 has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the IL-7 present in the sample binds to the capture antibody on the coated MP. 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-7 that has been captured onto the MP. 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-7 present in the sample when captured. The amount of IL-7 in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® IL-7 Immunoassay Prototype Assay1
Lower Limit of Detection 0.01 pg/mL
Lower Limit of Quantification2 0.05 pg/mL
Upper Limit of Quantification 56 pg/mL
Low-end CV% Range 2 - 9%
Low-end CV% Average 6%
Recommended Sample Volume 100 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-7] in a healthy reference


FIGURE 1. [IL-7] in EDTA plasma from 20 healthy donors, with median and interquartile range. Erenna® IL-7 Immunoassay Evaluation Reagent Kit reliably quantifies IL-7 in healthy subjects, who have a median [IL-7] of 0.85 pg/mL that is well above the detection limit of 0.01 pg/mL.

FIGURE 2. Erenna® IL-7 Immunoassay Evaluation Reagent Kit low-end standard curve signal (left) and curve fit (above).

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

Biology and Disease

Interleukin-7 (IL-7) is an endogenous cytokine responsible for the proliferation of B- and T-lymphocytes. The active human form of IL-7 has 152 aa and is expressed by fibroblasts, stromal, dendritic and endothelial cells. IL-7 often acts in synergy with other cytokines. For example, IL-7 is found to have an additive eff ect on T-cell activation in combination with IL-12. However, IL-7 is not redundant, as abnormal function of IL-7 has been implicated in immune disorders such as common variable immunodeficiency, HIV and rheumatoid arthritis (RA). Thus, IL-7 is an excellent candidate for therapeutic development because of its crucial role in the modulation of innate immunity. Both activation and suppression of IL-7 may be of value in future therapeutic interventions.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Interleukin-7
UniProtKB/Swiss-Prot ID: P13232
Protein-protein interaction database: P13232
Alternative Names:
Gene Names: IL7
Synonym Gene Names:
Function: Hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitors. It is important for proliferation during certain stages of B-cell maturation.
Biological Processes: bone resorption, cell-cell signaling, humoral immune response, organ morphogenesis, positive regulation of B cell proliferation, positive regulation of T cell differentiation
Molecular Function: cytokine activity, growth factor activity, interleukin-7 receptor binding

* Source Info from www.uniprot.org.


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