Selectin E/CD62e ELISA 试剂盒

If crystals form in the Buffer Concentrates, warm and gently stir them until completely dissolved. Washing Buffer (1×): Pour entire contents (50 ml) of the Washing Buffer (20×) into a clean 1,000 ml graduated cylinder. Bring to final volume of 1,000 ml with pure or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2 to 25℃. Washing Buffer (1×) is stable for 30 days. Assay Buffer (1×): Pour the entire contents (5 ml) of the Assay Buffer (10×) into a clean 100 ml graduated cylinder. Bring to final volume of 50 ml with distilled water. Mix gently to avoid foaming. Store at 2 to 8℃. Assay Buffer (1×) is stable for 30 days. Detect Antibody: Mix well prior to making dilutions. Make a 1：100 dilution of the concentrated Detect Antibody solution with Assay Buffer (1×) in a clean plastic tube as needed. The diluted Detect Antibody should be used within 30 minutes after dilution. Streptavidin-HRP: Mix well prior to making dilutions. Make a 1：100 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1×) ina clean plastic tube as needed. The diluted Streptavidin-HRP should be used within 30 minutes after dilution. Sample Dilution: If your samples have high sE-Selectin content, dilute serum/plasma samples with Assay Buffer (1×). For cell culture supernates, dilute with cell culture medium. Human sE-Selectin Standard: Briefly spin the vial at 6,000 rpm for 30 seconds before opening. Reconstitute Human sE-SelectinStandard by addition of distilled water. Reconstitution volume is stated on the label of the standard vial. Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted standard = 4,000 pg/ml). Allow the standard to reconstitute for 10 - 30 minutes. Mix well prior to making dilutions. Use polypropylene tubes. For serum/plasma samples, mixing concentrated human sE-Selectin standard (250 μl) with 250μl of Assay Buffer (1×) creates the high standard (2,000 pg/ml). Pipette 250 μl of Assay Buf er (1×) into each tube. Use the high standard to produce a 1:1 dilution series (scheme below). Mix eachtube thoroughly before the next transfer. Assay Buf er (1×) serves as the zero standard (0 pg/ml). For cell culture supernates, mixing concentrated human sE-Selectin standard (250 μl) with 250μl of cell culture medium creates the high standard (2,000 pg/ml). Pipette 250 μl of cell culture medium into each tube. Use the high standard to produce a 1:1 dilution series. Mix each tube thoroughly before the next transfer. Cell culture medium serves as the zero standard (0 pg/ml).

Bring all reagents and samples to room temperature before use.
1. Prepare all reagents including microplate, samples, standards and working solution as described in the previous sections.
2. Remove excess microplate strips and return them to the foil pouch containing the desiccant pack, and reseal for further use.
3. Add 300 μl Washing Buf er (1×) per well, and allow it for about 30 seconds before aspiration. Soaking is highly recommended to obtain a good test performance. Empty wells and tapmicrowell strips on absorbent pad or paper towel to remove excess Washing Buf er (1×). Use the microwell strips immediately after washing. Do not allow wells to dry.
4. Add 100 μl of 2-fold diluted Standard in duplicate. Add 100 μl of Standard Diluent to Blank well induplicate.
5. Add 90 μl of Assay Buf er (1×) and 10 μl sample to the sample well.
6. Add 50 μl of 1:100 diluted Detect Antibody to each well. Ensure reagent addition in step 4, 5and 6 is uninterrupted and completed within 15 minutes.
7. Seal the plate with a fresh adhesive film. Incubate at room temperature (18 to 25℃) for 2 hours on a microplate shaker set at 300 rpm.
8. Aspirate each well and wash by filling each well with 300 μl Washing Buf er (1×), repeat five times for a total six washes. Complete removal of liquid at each step is essential to the best performance. After the last wash, remove any remaining Washing Buf er (1×) by aspirating or decanting. Invert the plate and tap it against clean paper towels.
Human sE-Selectin ELISA Kit
9. Add 100 μl of diluted Streptavidin-HRP to each well.
10. Seal the plate with a fresh adhesive film. Incubate at room temperature (18 to 25℃) for 45minutes on a microplate shaker set at 300 rpm.
11.Repeat aspiration/wash as in step 8.
12.Add 100 μl of Substrate Solution to each well. Incubate for 10 - 30 minutes at roomtemperature. Protect from light.
13.Add 100 μl of Stop Solution to each well. The color will turn yellow. If the color in the well is green or if the color change does not appear uniform, gently tap the plate to ensure thoroughmixing.
14. Measure the optical density value within 30 minutes by microplate reader set to 450 nm. If wavelength correction is available, set to 570 nm or 630 nm. If wavelength correction is not available, subtract readings at 570 nm or 630 nm from the readings at 450 nm. This subtractionwill correct for optical imperfections in the plate. Reading directly at 450nm without correction may generate higher concentration than true value.

Average the duplicate optical density readings for each standards and sample, then subtract the average optical density value of the zero standard. Standard Concentration as horizontal axis, optical density (OD) Value as the vertical axis, regressing the data and create a standard curve using computer software. The data may be linearized by plotting the log of the sE-Selectin concentrations versus the log of the OD and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. Note: The finally concentration of top standard is 6000 pg/mL. If instruction in this protocol have been followed samples have been diluted by 1:1 ratio (50 μL sample + 50 μL Assay Buffer), the concentration read from the standard curve must be multiplied by the dilution factor (x2). If serum/plasma samples have been diluted following the instruction, the final dilution factor is 10.