ELISA Kit for the Quantitative Analysis of GDF-8

ELISA Kit for the Quantitative Analysis of GDF-8

 

The human GDF-8 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human GDF-8 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 

Growth Differentiation Factor 8 (GDF-8), also known as Myostatin, is a secreted TGF-β superfamily protein. GDF-8 is synthesized as a 376 amino acid (aa) preproprotein that consists of a 24 aa signal peptide, a 243 aa propeptide, and a 109 aa mature protein. The secreted proprotein is cleaved by BMP-1 family proteases to separate the 35-40 kDa propeptide from the 12 kDa bioactive mature protein. Within the propeptide, mouse GDF-8 shares 96% and 99% aa sequence identity with human and rat GDF-8, respectively. The amino acid sequence of mature GDF-8 is 100% conserved between human, mouse, and rat.

Myostatin is expressed in developing and adult skeletal muscle. Myostatin is a potent negative regulator of skeletal muscle mass, Myostatin can control smyoblast proliferation and upregulate in cardiomyocytes surrounding infarcted areas after mycardial infarction. Myostatin is released by mechanically-stressed cardiomyocytes and induces the skeletal muscle wasting which is common in heart failure. Genetic GDF-8 inactivation or blockade leads to multiple changes in energy balance and metabolism including increased fatty acid oxidation and brown fat formation, reduced white fat accumulation and circulating cholesterol and triglyceride levels, and increased peripheral insulin sensitivity and resistance to diet-induced obesity.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human GDF-8. An anti-human GDF-8 monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human GDF-8 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human GDF-8 biotin-conjugated antibody were added and binds to human GDF-8 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 GDF-8 in the original specimen.  

Materials provided with the kits:

Reagent	96/48Test Kit
GDF-8Antibody-Coated Wells	12strips/6strips
Standard Diluent	10ml/5ml
GDF-8 Standard	4/2vial(s)
GDF-8 Detection Antibody	10ml/5ml
Streptavidin-HRP	10ml/5ml
Wash Buffer Concentrate 20×	30ml/15ml
TMB	10ml/5ml
Stop Solution	5ml/3 ml
Plate Covers	3/2
Complete Instruction Manual	1

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 diluted 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 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 26-28℃.

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. Dilute 1ml of 5×sample diluent into 4ml deionized or distilled water to 1×sample diluent.

4. Add the standard dilution solution 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 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 range in 2 ~ 10 times, if there is no definite scope, advice from 4 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

GDF-8 Standard Curve

 

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was29.4pg/ml.

Specificity: No significant cross-reactivity or interference with human Activin RIIB/Fc Chimera，GASP-2，GDF-11

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