Package size: 96 determinations                           Catalogue No.: QRCT –561216EIA \ UTL

Storage: 2 - 8 °C

I. INTRODUCTION

Interleukin 6 (IL-6) is a multifunctional protein produced by lymphoid and non-lymphoid cells, and by normal and transformed cells, including T cells, monocyte/macrophages, fibroblasts, hepatocytes, vascular endothelial cells, cardiac myxomas, bladder cell carcinomas, myelomas, astrogliomas and glioblastomas. The production of IL-6 in these various cells is regulated, either positively or negatively, by a variety of signals including mitogens, antigenic stimulation, lipopolysaccharides, IL-1, TNF, PDGF and viruses. On the basis of its various activities, IL-6 has also been called interferon-β2 (IFN-β2), 26 kDa protein, B-cell stimulatory factor-2 (BSF-2),　hybridoma/plasmacytoma growth factor, hepatocyte stimulating factor, cytotoxic T-cell differentiation factor, and macrophage-granulocyte inducing factor 2A (MGI-2A).The IL-6 cDNA sequence predicts a protein of 212 amino acid residues in length with two potential N-glycosylation sites. The hydrophobic N-terminal 28 amino acid residue signal peptide is cleaved to produce a mature protein of 184 amino acids with four cysteine residues and a predicted molecular mass of 21 kDa. Mouse IL-6 cDNA sequence shows a homology of 42% at the amino acid level when compared with the human sequence. Sequencing of the genomic DNA for IL-6 indicates that the gene for this factor consists of five exons and four introns. On the basis of sequence similarity and gene structural motif similarity, IL-6 can be grouped in a family of cytokines that also includes OSM, G-CSF, LIF, and CNTF. All of these cytokines are predicted to have a four helix bundle structure similar to that found for growth hormone, suggesting that they all evolved from a common ancestral gene.

II. REAGENTS

Materials provided with the kits :

1、Coated Microtitration Strips 96 wells.

2、Standard  5.0,  10,  20,  40 ，80pg/ml, 1 set.

、Enzyme Conjugate Solution, 12 ml.

、Substrate  A, 6 ml.

、Substrate  B, 6 ml.

、Stopping Solution (1N HCl), 6 ml.

、Rinsing buffer,60 ml( x 20).

、dilution,15ml(x5)

Materials required but not provided :

Precision pipettes: 0.10, 0.20, and 1.0 ml.

???Disposable pipette tips.

???Distilled water.

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        FOR INFORMATIONAL USE ONLY ? DO NOT USE FOR PERFORMING ASSAY ? REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT

 goat anti –Rabbit Interleukin-6

                                                         Collect Sample – serum or blood plasma

Package size: 96 determinations                           Catalogue No.: QRCT –561216EIA \ UTL

Storage: 2 - 8 °C

?? Vortex mixer or equivalent.

?? Absorbent paper or paper towels.

?? Microtiter plate shaker

?? Graph paper.

?? A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450 nm .

III. STORAGE OF TEST KIT

Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as described above. A  microtiter  plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. All reagents should be allowed to reach room temperature before use.

IV. ASSAY PROCEDURE

STEP1     Prepare the ringing buffer by diluting the ringing buffer 20×(example: 60ml+1140ml H2O), Prepare the Sample Diluent by diluting the Diluent 5×(example: 15ml+60ml H2O)

STEP2     Dilute samples 1:100 distribing 5ul of sample into 500 ul of dilluent .

STEP3    Place 100ul of diluted sample /reagent blank/standard/controls to the wells of the strips. Incubate for 30 min at 37℃。Wash 5 times。

STEP4    Add 100ul of  Enzyme Conjugate to each well。 Incubate for 30 min at 37℃。Wash 5 times。

STEP5    Add 50ul Substrate A and Substrate B to each well。Incubate for 15 min at 37℃。

STEP6    Add 50ul of stop solution。read absorbance at 450nm within 15 min

V. Important Notes

The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. It is recommended that if manual pipetting is used, no more than 32 wells be used for each assay run, since pipetting of all standards, specimens and controls should be

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FOR INFORMATIONAL USE ONLY ? DO NOT USE FOR PERFORMING ASSAY ? REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT

 goat anti –Rabbit Interleukin-6

                                                          Collect Sample – serum or blood plasma

Package size: 96 determinations                           Catalogue No.: QRCT –561216EIA \ UTL

Storage: 2 - 8 °C

 completed within 3 minutes. A full plate of 96 wells may be used if automated pipetting is available. Duplication of all standards and specimens, although not required, is recommended.

VI. CALCULATION OF RESULTS

Calculate the average absorbance values (A450) for each set of reference standards, control, and samples. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration on linear graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis.Using the mean absorbance value for each sample, determine the corresponding concentration of in from the standard curve.

VII. EXAMPLE OF STANDARD CURVE

Results of a typical standard run with optical density readings at 450nm shown in the Y axis against concentrations shown in the X axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve.

VIII. CALCULATION OF RESULTS

The minimum detectable concentration of IL-6 in this assay is estimated to be 0.5 pg/ml.

IX. LIMITATIONS OF THE PROCEDURE

Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. The results obtained from the use of this kit should be used only as an adjunct to other diagnostic procedures and information available to the physician.

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