ELISA Kit for the Quantitative Analysisof Human CD138

The human CD138ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human CD138in 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

CD138，also known as Syndecan-1,is a type I membrane protein that belongs to the syndecan family of Type 1 transmembrane proteins. Its precursor is a 310 amino acid (aa) protein with a 17 aa signal sequence, a 234 aa extracellular domain (ECD), a 25 aa TM segment, and a 34 aa cytoplasmic region that includes a PDZ binding motif with a tyrosine phosphorylation site .Human CD138 ECD is 65 - 71% identical to the ECD of rat, mouse, canine, equine and bovine CD138 at aa level.

CD138is primarily expressed on epithelial cells and terminally differentiated B cells.TheCD138 protein functions as an integral membrane protein, participating in cell proliferation, cell migration and cell-matrix interactions via its receptor for extracellular matrix proteins. It can bind chemokines to create chemotactic gradients when shed, and also binds integrins to modulate the influx of leukocytes .Altered CD138 expression has been detected in several different tumor types. Some researches have found that shedding of CD138 can facilitate growth, angiogenesis and metastasis in myeloma and other cancers.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human L -Selectin. An anti-human CD138 monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human CD138 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human CD138 biotin-conjugated antibody were added and binds to human CD138 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 CD138 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 Serumsamples.

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 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 deionized or distilled water to the bottle according to the volume of the label and wait 15 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 into 300ul wash buffer and soak 15 or 30seconds,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.The samples or different concentrations of standard products were added to the corresponding holes according to 100μl/ well, and the reaction holes were sealed with sealing plate film, and incubated at room temperature (25-28℃) for 120 minutes. For different samples of serum plasma, the dilution ratio is different, the dilution times are generally 5 to 120 times dilution, if there is no accurate dilution range, it is recommended to dilute with 1× standard diluent from 10 times.

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 CD138 standard curve

 

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 114.5pg/ml.

Specificity: No significant cross-reactivity or interference with human Eotaxin, GROa , HGF , MCP-3 , Midkin , MMP-7 , RANTES , Syndecan-2 , Syndecan-3 , Syndecan-4 , TARC and Mouse Syndecan-1.

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