Human granulocyte colony stimulating factor ( G-CSF ) ELISA kit
Japan IBL imported, article number: 27131
Japan's IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
Introduction
G-CSF belongs to the class of cytokines that stimulate the differentiation and proliferation of neutrophil precursor cells. It is produced by fibroblasts and vascular endothelial cells, and it has been reported that it can stimulate proliferation in some tumors. Human G-CSF kit detects human G-CSF
principle
This kit uses two different highly specific antibodies, TMB as a colorant according to the sandwich principle of enzyme-free, and the color intensity is proportional to the amount of human G-CSF.
examination range
7.81~500pg/ml (1 hour incubation at 37°C for the first time)
1.95~250pg/ml (first incubation at 4°C overnight): internal data
expected usage
1. This kit is used for quantitative detection of human G-CSF in cell supernatants.
2. Both recombinant and natural G-CSF can be detected with this kit.
3. Experience has shown that lower levels of dilution of serum or EDTA plasma may result in lower results due to interference with whole blood (see Appendix I).
Kit composition
1. Coated plate, 96-well, coated with anti-human G-CSF mouse IgG monoclonal antibody, affinity purification
2. Labeled antibody concentration, 30X, 0.4mlx1, HRP-labeled anti-human G-CSF rabbit IgG Fab', affinity purification
3. Standard, 1.0mlx2, recombinant human G-CSF
4. EIA buffer, 30mlx1, 1% BSA, PBS containing 0.05% Tween 20
5. Label antibody dilution, 12mlx1, 1% BSA, PBS containing 0.05% Tween 20
6. Colorant, 15mlx1, TMB substrate solution
7. Stop solution, 12mlx1, 1N sulfuric acid
8. Wash buffer concentrate, 50mlx1, 40X, phosphate buffer containing 0.05% Tween 20
Materials required for the experiment but not provided in the kit
1. Microplate reader (450nm)
2. Pipettes and tips
3. Beaker
4. Distilled water
5. Incubator (37 ± 1 ° C)
6. Coordinate paper (logarithm-logarithm)
7. Absorbent paper
8. Standard dilution tube
9. Wash the bottle
10. Disposable test tube
ready
1. Preparation of washing buffer: first equilibrate the washing concentrate to room temperature, mix well, then mix 50 ml of the concentrated solution with 1950 ml of deionized water to obtain. The prepared washing solution should be stored in the refrigerator and used up within 2 weeks.
2. Preparation of labeled antibody: The labeled antibody was diluted 30-fold with a labeled antibody dilution to obtain.
Example: If only one plate is measured, 8 wells, you need to label the antibody 800ul (30ul of labeled antibody + 870ul of labeled antibody dilution, mix, add 100ul per well)
This step is completed before the experiment
The remaining labeled antibody concentrate should be stored in a sealed bottle and stored at 4 ° C
3. Preparation of standard product: Pipette 1.0ml of deionized water into the standard bottle and mix well to obtain 1000gg/ml human G-CSF standard.
4. Dilution of standard: Prepare 8 tubes for standard dilution, add 230 ul EIA buffer to each tube
The concentration of each tube is as follows
No. 1 tube 500pg/ml
Tube 2 250pg/ml
Tube No. 3 125pg/ml
Tube 4 62.5pg/ml
Tube 5 31.25pg/ml
No. 6 tube 15.63pg/ml
Tube 7 7.81pg/ml
Tube 8 0pg/ml (test sample blank)
Pipette 230 ul of the standard product into the No. 1 tube, then pipette 230 ul from the No. 1 tube into the No. 2 tube and serially dilute it. The standard range is 500~7.81 ng/ml.
Internal experimental procedure ( incubated overnight at 4 °C )
High-sensitivity steps necessary
1) Preparation of standard product: Pipette 2.0ml of deionized water into the standard bottle and mix well to obtain 500gg/ml human G-CSF standard.
2) Dilution of standard: Prepare 9 tubes for standard dilution, add 230 ul EIA buffer to each tube
The concentration of each tube is as follows
No. 1 tube 250 pg/ml
Tube 2 125pg/ml
Tube 3 62.5pg/ml
Tube 4 31.25pg/ml
No. 5 tube 15.63pg/ml
No. 6 tube 7.81pg/ml
Tube No. 7. 3.91pg/ml
Tube 8 1.95pg/ml
Tube 8 0pg/ml (test sample blank)
Pipette 230 ul of standard product into the No. 1 tube and mix it. Then take 230 ul from the No. 1 tube and add the No. 2 tube, and serially dilute it. The standard range is 250~1.95pg/ml. The No. 9 tube is the test sample blank.
5. Sample dilution: Samples were diluted in EIA buffer. If the sample concentration has not been established beforehand, we recommend that the sample be tested with several different dilution ratios to determine the optimal dilution ratio of the sample.
Experimental procedure
Before use, all samples should be equilibrated to room temperature for about 30 minutes, then thoroughly mixed to ensure no change in reagent quality. Standard curve and sample detection are performed simultaneously.
Figure:
1. Add 100ul EIA buffer to Reagent Blank
2. Add a test sample blank, add 100 ul of sample and diluted standard to the corresponding well; (internal procedure)
3. Cover plate, incubate for 1 hour at 37 ° C (incubated overnight at 4 ° C)
4. Wash the plate, repeat 7 times or more, pat dry on the absorbent paper; wash the plate to wash the plate 4 times
5. Add 100 ul of labeled antibody solution to each well, except for Reagent Blank
6. Cover plate, incubate for 30 min at 37 °C
7. Wash the plate 9 times, same as step four
8. Pipette the TMB substrate solution required for the test into a disposable tube and add 100 ul of TMB substrate solution to each well. The remaining TMB substrate fluid can no longer be returned to the original bottle to avoid contamination
9. In the dark, incubate for 30 min at room temperature, the solution turns blue
10. Add 100 ul of stop solution to each well and the solution turns blue.
11. Wipe off the stain or water droplets at the bottom of the microplate. Within 30 minutes after the stop solution is added, read at 450nm.
pay attention
1. The test sample should be tested immediately after collection. The frozen sample should avoid repeated freezing and thawing. It should be completely dissolved and mixed before testing.
2. Test sample diluted with EIA buffer
3. Test samples and standards should be double tested
4. The test sample should be in the neutral pH range, and the contamination of organic solvents may affect the detection.
5. Only use the washing liquid provided by the kit to wash the plate, otherwise the test will fail.
6. Dry the microplate on the absorbent paper, do not wipe
7. The TMB substrate solution should be stored in the dark as it is sensitive to light and avoids contact with metals.
8. The microplate reader must be read within 30 minutes after the addition of the stop solution.
Result calculation
Before plotting the curve, all data should be subtracted from the test sample blank values, including standards and unknown samples. Draw a standard curve on the log-logarithmic scale paper with the OD value and concentration of the standard, and directly read the concentration of the unknown sample from the standard curve.
Attachment: Using automatic microplate reader detection, only need to set the relevant parameters of the microplate reader before the detection, such as coordinate parameters (linear, semi-logarithmic) and calculation mode parameters (three regression, four parameters or double logarithmic curve equation) , you can get the results directly
Appendix I:
This translation is for reference only, please refer to the original for details.
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