ELISA Kit for the Quantitative Analysis of Human HEPCIDIN

The human HEPCIDIN ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human HEPCIDIN in cell culture supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY.

Introduction

Interleukin-4 is a pleiotropic cytokine, mainly produced by activated T-lymphocyte cells, mast cells and bone marrow stromal cells. HEPCIDIN exhibits in molecular weight forms between 15 and 19 kDa based on variable glycosylation.HEPCIDIN regulates the function of the immune response in variety of cells. HEPCIDIN participates in at least several B-cell activation processes as well as other cell types. It is a costimulator of DNA-synthesis. It induces the expression of class II MHC molecules on resting B-cells. It enhances both secretion and cell surface expression of IgE and IgG1; it also regulates the expression of the low affinity Fc receptor for IgE (CD23) on both lymphocytes and monocytes.                                                              

HEPCIDIN plays an important role in regulating the subtype Conversion of antibodies, it is also the important regulator involved in the differentiation of Th cell precursor into Th2 cells, and then indirectly regulates the humoral immune response and the generation of antibodies. In addition, genetic variations in IL4 may be a cause of susceptibility to ischemic stroke , cerebrovascular accident or cerebral infarction.

Principles of the Test

The kit is a solid sandwich enzyme-linked immunosorbent assay for detection of human HEPCIDIN. An anti-human HEPCIDIN monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human HEPCIDIN in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human HEPCIDIN biotin-conjugated antibody were added and binds to human HEPCIDIN captured by the first antibody, which formed a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be removed during a wash step. After this, substrate solution would be added and catalyzed by the HRP, and a coloured product is formed. The intensity of the colored product is used to calculate in proportion to the amount of human HEPCIDIN in the original specimen.

Materials provided with the kit:

Specimen Collection

1. Collecting specimen as following:

A. The particulate of the cell culture supernatants should be removed before use.

B. Serum was obtained from clot at room temperature.

C. Please collect plasma with EDTA.

D. Assay immediately or store samples at -20℃. Avoid free-thaw cycles.

2. Antiseptic and anticoagulant should not appear in Serum

samples.

3. Any particulate should be removed from samples before use.

4. Do not use grossly hemolyzed or lipemic samples.

Precautions for use:

1.Please storage the Kit at 2～8℃。

2. Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use.

3. Please discard the remains after use of the dissolved standard.

4. Avoid contact of substrate solution with oxidizing agents and metal.

5. Usage of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens.

6. Do not mix or substitute reagents with those from other lots or other sources.

7. To ensure the adequate mixture of added reagents, please tap gently the plate after the wells were filled with liquid.

8. Incubation temperature should be 25～28℃.

9. Wash step was crucial for whole assay process.

10. Duplicate wells of the same sample were recommended in

assay process.

11. Avoid the foam while pour the liquid into wells.

12．For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.

Reagent Preparation

1.The reagents should be warmed up to room temperature before use.The remanent reagents must reseal and put into refrigeratory again as soon as possible.

2.Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work.

3. If you have a 5x standard diluent, please dilute it with double steaming water or deionized water.

4. Add standard diluent to the bottle according to the volume of  the label and wait15 minutes for complete dissolution. Incubation temperature should be 25- 28℃。.And in turn add the half concentration diluent by standard diluent .

Wash step:

Automated microplate washer or operating by pipette: Each well should be pour into300ul wash buffer and soak 15 or 30 seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by aspirating. Invert the plate and blot it against clean paper towels.

Materials Required But Not Provided

1. pipettes and pipette tips

2. Microwell strip reader capable of reading at 450 nm (540 nm as optional reference wave length)

3. automated microplate washer

4.Glass-distilled or deionized water

Assay procedure

1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.

2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient density of standard for standard curve.

3.Add 100ul of standard or sample.Cover with the Plate Covers provided.Incubate for 2 hours at room temperature If is plasma serum samples, different sample dilution ratio is different, generally in the range of 100 ~ 1000 times, if there is no definite scope, advice from 200 times dilution, if the sample concentration is too high, more than test scope, please increase after diluted times dilution test again.

4.Five times wash process were repeated.

5.Add 100ul of detection antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature.

6.Five times wash process were repeated.

7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature.

8.Five times wash process were repeated.

9.Add 100ul of TMB，Lucifugal incubation for 20 minutes at room temperature.

10.Add 50ul of stop solution to each well, determine the optical

density of each well within 10 minutes.

Calculation of Results

1.Duplicates should be within 20 percent of the mean. Average absorbance values for each set of duplicate samples were used as detection results.

2.The blank absorbance values of subtract should be deducted.

3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD value (on the Y axis) vs. concentration (on the X axis). The sample concentration was obtained based on its OD value founding in the standard concentration curve.

4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again

Typical Data and Standard Curve

Human HEPCIDIN Standard Curve

 

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 3.2pg/ml.

Specificity : : No significant cross-reactivity or interference with human α2-Macroglobulin and mouse hepcidin. This Kit recognizes human Hepcidin-20, human Hepcidin-22,human Hepcidin-24, and human Hepcidin-25.

Repeatability: The coefficient of variation between wells or plates is less than 10 percent.