ELISA Kit for the Quantitative Analysis of Human GRANZYME B

The human GRANZYME B ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human GRANZYME B in cell culture supernatants,human serum and plasma.

Introduction

Granzyme B, one of the five Granzymes (A, B, H, K and M) identified in the human genome, belongs to the granzyme family of serine proteases. Granzyme B is a 32kDa protein, which is specifically found in granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Synthesized as a precursor (247 residues) with a signal peptide(residues 1-18), a pro-peptide (residues 19-20), and a mature chain (residues 21-247) , it is fully processed into the mature chain and becomes anactive protease when the pro-peptide, Gly-Glu is removed from the N-terminus. Respectively , Granzyme B is 71%, 69%, and 68% identical to its canine, rat, and mouse counterparts. 

Granzyme B plays an essential role in granule-mediated apoptosis.It has been shown to be an important mediator of skin injury, repair and inflammation through extracellular substrates including VE-Cadherin , Laminin and Fibronectin etc. Different from Granzyme A plasma levels which remain unchanged, Granzyme B plasma levels have been found higher with atopic dermatitis and psoriasis when compared to healthy controls. Serum from patients with Crohn’s disease also have significantly higher Granzyme B levels than controls .These all strongly indicate the effect of Granzyme B in modulating inflammation.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human GRANZYME B. An anti-human GRANZYME B monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human GRANZYME B in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human GRANZYME B biotin-conjugated antibody were added and binds to human GRANZYME B 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 GRANZYME B 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 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 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. Add 0.5 ml standard diluent to bottle 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 300 ul 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, Each hole add buffer 50 ul sample analysis, add in 1 x standard sample after 5 times diluent dilution, and sample amount to 50 ul.

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  Granzyme B Standard Curve

 

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 17.5pg/ml.

Specificity: No significant cross-reactivity or interference with FGF R1β(IIIc)/Fc Chimera,Granzyme A,Granzyme H.

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