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Immunoassays | Inflammation & Cytokines

ErennaŽ IL-12 Immunoassay Kit (Cat# 03-0057-xx)

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Introduction

The ErennaŽ IL-12 Immunoassay uses a quantitative fluorescent sandwich immunoassay technique to measure IL-12 in human serum and plasma samples. A capture antibody specific for human IL-12 has been pre-coated onto paramagnetic microparticles (MPs). The user pipettes MPs, standards, and samples into uncoated microplate wells. During incubation, the IL-12 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-12 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 ErennaŽ 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-12 present in the sample when captured. The amount of IL-12 in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the ErennaŽ IL-12 Immunoassay Kit1
Lower Limit of Detection 0.01 pg/mL
Lower Limit of Quantification2 0.05 pg/mL
Upper Limit of Quantification 12.5 pg/mL
Low-end CV% Range 2 - 8%
Low-end CV% Average 4%
Recommended Sample Volume 100 μL
Minimum Sample Volume Required3 38 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ą 20% recovery
3 based upon median [IL-12] in a healthy reference


FIGURE 1. [IL-12] in EDTA plasma from 10 healthy donors, with median and interquartile range. The ErennaŽ IL-12 Immunoassay Evaluation Reagent Kit reliably quantifies IL-12 in healthy subjects, who have a median [IL-12] of 0.13 pg/mL that is well above the detection limit of 0.01 pg/mL.

FIGURE 2. ErennaŽ IL-12 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-12 (IL-12) is a cytokine expressed by T-cells and natural killer cells in response to the presence of microbial agents. Increased expression of IL-12 drives a positive feedback loop, which generates more T-cells and natural killer cells, along with other immuno-regulatory cytokines such as IFN-γ, IL-10 and TNFα. Although IL-12 is an important initiator of cell-mediated immunity, its pro-inflammatory effects can be deleterious. Over production of this cytokine has been implicated in several autoimmune disorders such as inflammatory bowel disease, psoriasis, and Crohn’s disease. The ability to escalate and strengthen the immune response makes IL-12 an important immunostimulant therapy for infectious diseases and metastatic cancers.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Interleukin-12 receptor subunit beta-1
UniProtKB/Swiss-Prot ID: P42701
Protein-protein interaction database: P42701
Alternative Names: IL-12 receptor beta component, CD_antigen=CD212
Gene Names: IL12RB1
Synonym Gene Names: IL12R, IL12RB
Function: Functions as an interleukin receptor which binds interleukin-12 with low affinity and is involved in IL12 transduction. Associated with IL12RB2 it forms a functional, high affinity receptor for IL12. Associates also with IL23R to form the interleukin-23 receptor which functions in IL23 signal transduction probably through activation of the Jak-Stat signaling cascade.
Biological Processes: positive regulation of cell proliferation
Molecular Function: cytokine receptor activity

* Source Info from www.uniprot.org.


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