Interleukin-6 (IL-6) is a multifunctional cytokine with an α-helical structure, a molecular weight of 22- 28 kD, and varying degrees of phosphorylation and glycosylation. It plays a significant role in various aspects of diseases, including acute-phase responses, inflammation, hematopoiesis, bone metabolism, and cancer progression. The production of IL-6 is typically controlled by glucocorticoids, catecholamines, and second-class steroids and is generally associated with cellular activation. In the blood of normal individuals, IL-6 levels are within the range of 1 pg/mL, with slight increases during the menstrual cycle, moderate increases in the late stages of certain cancers, and significant increases after major surgery. IL- 6 triggers cellular signaling through cell surface receptors, which are composed of a ligand-binding subunit (IL-6 receptor) and a signal transduction subunit gp130, forming a heterodimeric complex. IL-6, in conjunction with tumor necrosis factor-alpha (TNF-α) and IL-1, plays a nearly unique role in the acute inflammatory response, including fever and acute liver inflammation. It also plays a crucial role in the transition from acute inflammation to acquired immunity or chronic inflammatory diseases. Dysregulation of IL-6 can promote chronic inflammation, such as obesity, insulin resistance, inflammatory bowel disease, inflammatory arthritis, and sepsis.

This assay kit employs the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) technique. Specific anti-mouse IL-6 antibodies with high affinity are pre-coated onto the enzyme-linked plate. In the wells of the enzyme-linked plate, standards, test samples, and biotinylated detection antibodies are added. After incubation, IL-6 present in the samples binds to the solid-phase antibody and the detection antibody, forming an immune complex. After removing unbound substances, streptavidinhorseradish peroxidase labeled with horseradish peroxidase (HRP) is added. After wash step to remove any unbound substances, 3,3′,5,5′-Tetramethylbenzidine (TMB)substrate is added and color develops in proportion to the amount of IL-6 bound initially. The assay is stopped, and the optical density of the wells is determined using a micro-plate reader at 450nm wavelength (with a reference wavelength of 540nm or 570nm) for quantification.

Recovery
Mouse IL-6 of different levels was spiked into cell culture medium samples to determine its recovery rate. The recovery rate ranged from 70-114%, with an average recovery rate of 91%.
Sensitivity
The minimum detectable dose (MDD) of mouse IL-6 is typically less than 1.8 pg/mL.
The MDD was determined by adding two standard deviations to the mean O.D. value of twenty zero standard replicates and calculating the corresponding concentration.
Linearity
To assess the linearity of the assay, four samples spiked with high concentrations of mouse IL-6 were diluted with 1×Reagent Diluent to bring them within the detection range.

Specificity
This assay recognizes natural and recombinant mouse IL-6.

The factors listed below were prepared at 50 ng/mL in 1×Reagent Diluent and assayed for cross-reactivity.

Preparations of the following factors prepared at 50 ng/mL in a mid-range mouse IL-6 control were assayed for interference. No significant cross-reactivity or interference was observed.