ELISA Kit for the Quantitative Analysis of Human CXCL7

The human CXCL7 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human CXCL7 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

Chemokine (C-X-C motif) Ligand 7 (CXCL7), also known as neutrophil activating peptide 2 (NAP-2),is a platelet-derived growth factor that belongs to the CXC chemokines containing an ELR domain (Glu-Leu-Arg tripeptide motif).It shares the same CXC chemokine domains with connective Tissue Activating Protein III (CTAPIII),β thrombogulin (βTG) and platelet basic protein (PBP), which represent amino-terminal extended variants of CXCL7.However, these three proteins do not exhibit CXCL7/NAP2 activity .Similar to other ELR domain containing CXC chemokines, CXCL7 binds CXCR2,which is the chemoattr actants and activators for neutrophils.CXCL7 has also been shown to stimulate various cellular processes, such as DNA synthesis , mitosis, glycolysis, intracellular cAMP accumulation, prostaglandin E2 secretion ,etc.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human CXCL7. An anti-human CXCL7 monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human CXCL7 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human CXCL7 biotin-conjugated antibody were added and binds to human CXCL7 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, subtrate 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 CXCL7 in the original specimen.

Materials provided with the kits:

Reagent	96/48Test Kit
Human CXCL7 Antibody-Coated Wells	12 strips/6 strips
5XStandard Diluent	20ml/10ml
Human CXCL7 Standard	2/1vial(s)
Human CXCL7 Detetion 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 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 mixure 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.

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 be diluent 1000~20000 times before add into the wells, If there is no definite scope, it is suggested that since 2000 times, 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 detetion 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 valure (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

concentration (pg/ml)	Typical data 1	Typical data 2	Average
0	0.122	0.091	0.1065
31.25	0.284	0.194	0.239
62.5	0.398	0.363	0.3805
125	0.652	0.504	0.578
250	1.168	1.01	1.089
500	1.923	1.806	1.8645
1000	3.053	2.869	2.961

Human CXCL7 Standard Curve

 

Sensitivity,Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 14.5pg/ml.

Specificity : No Cross Reactivity with human BLC , IP-10 , ENA-70 , ENA-74 , ENA-78 ,  MIG , GROa , GROb , SDF-1a , IL-8.

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