ELISA Kit for the Quantitative Analysis of Human DKK-1

The human DKK-1 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human DKK-1 in cell culture supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY.Please read this instruction manual carefully and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call us for other aim.

Introduction

Dickkopf-1 is the first identified member of the Dickkopf family and was discovered in Xenopus head as an inducer. Dkk proteins include two cysteine-rich structural domain,and the C-terminal domain has 10 cysteine residue that can help protein to fold, playing a necessary and essential role in Wnt inhibition. Mature Human Dkk-1 is a 40kDa glycoprotein that shows 86%, 87%, 91% and 90% amino acid (aa) sequenceidentity with mouse, rat, bovine, and rabbit Dkk-1, respectively. In adult, Dkk-1 is expressed mainly by osteoblasts and osteocytes

Postnatally, Dkk-1 is expressed mainly by osteoblasts and osteocytes. High Dkk-1 expression has been shown and may be pathogenic in conditions wherebone is eroded, such as rheumatoid arthritis, multiple myeloma, Paget’s disease, andglucocorticoid-induced osteoporosis. Dkk-1 also regulates skin pigmentation and thickness by controlling Wnt signaling inmelanocytes. Activation of Wnt by repression of Dkk-1 activity may be a factor inoncogenic transformation, for example, by the oncogene c-mycin mammary epithelial celltransformation or in human colon cancer. Dkk-1 is also expressed in platelets. Release of platelet Dkk-1 occurs during activation ofplatelets, including clotting during collection of serum samples. Dkk-1 has been hypothesized toplay a role in platelet-mediated endothelial cell activation leading to plaque formation.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human DKK-1. An anti-human DKK-1 monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human DKK-1 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human DKK-1 biotin-conjugated antibody were added and binds to human DKK-1 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 DKK-1 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.

Note: Strongly recommend that the serum and plasma samples should be diluent as doubling dilution before use.

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 theremains after useof 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 1.5ml 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 asoptional 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 temperatureIf assay the serum sample,you should add 50μl assay buffer with 50μl sample into the wells,if the protein concentration is higher than the range of the Kit, add the same quantitys assay buffer with the sample, the deficiency should be complemented with sample diluent to 100μl per well.

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

TypicalData and Standard Curve

Human DKK-1 Standard Curve

 

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 57.9pg/ml.

Specificity: No significant cross-reactivity or interference with humanDkk-4, Kremen-1, Kremen-2, LRP-6.

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