DESCRIPTION

Interleukin-6 (IL-6) is a pleiotropic cytokine produced by various cells during infection, trauma, and immunological challenges. IL-6 acts via a receptor complex consisting of two distinct membrane-bound glycoproteins, an 80-kDa IL-6 receptor α (IL-6Rα) subunit (CD126, gp80) and a 130-kDa signal-transducing element (gp130, CD130). After binding of IL-6 to membrane-bound IL6R alpha, the complex of IL-6 and IL-6Rα associates with gp130, thus activating the receptor. Expression of gp130 is found in almost all organs while IL-6R alpha is predominantly confined to hepatocytes and leukocyte subpopulations (monocytes, neutrophils, T cells, and B cells). IL-6Rα is produced by proteolytic shedding and alternative splicing to produce soluble IL-6Rα.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	1,972.3	26.0	1.3
2	8	470.7	6.1	1.3
3	8	236.2	4.4	1.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,984.8	65.7	3.3
2	16	480.1	11.9	2.5
3	16	235.3	5.2	2.2

RECOVERY

The recovery of mouse IL-6R alpha spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:64	122	120-125
	1:128	118	108-123
Cell culture supernatant	1:2	120	112-130
	1:4	103	93-114

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concentmouseions of mouse IL-6R alpha and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Mouse serum samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The mouse serum was initially diluted 1:8. )

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	119
	Range (%)	-	115-123
1:4	Average% of Expected	103	114
	Range (%)	101-106	113-115
1:8	Average% of Expected	106	110
	Range (%)	105-106	104-115
1:16	Average% of Expected	86	109
	Range (%)	81-91	106-112

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse IL-6R alphain produced at Sunlong Medical™.