Osteopontin ELISA 试剂盒

Validation data Sensitivity and Limit of Detection Sensitivity was calculated by comparing the OD's for twenty wells run for each of the zero and standard #6. The detection limit was determined at two (2) standard deviations from the zero along the standard curve. Sensitivity was determined as 0.246 ng/mL. The Limit of Detection for the assay was determined in a similar manner by comparing the OD's for twenty replicates for each of the zero standard and a low concentration human urine sample. Limit of Detection was determined as 0.248 ng/mL.

reagent PreParation Allow the kit reagents to come to room temperature for 30 minutes. We recommend that all stan- dards and samples be run in duplicate to allow the end user to accurately determine Osteopontin concentrations. Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit. Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water. Once diluted this is stable at 4 °C for 3 months. Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water. Once diluted this is stable at room temperature for 3 months. Standard Preparation Add 500 μL of Assay Buffer to one vial of OPN standard to generate a 40 ng/mL Stock. Label six test tubes as #1 through #6. Pipet 250 μL of Assay Buffer into tubes #1 to #6. Add 250 μL of the 40 ng/mL Osteopontin stock solution to tube #1 and vortex completely. Take 250 μL of the Osteopontin solution in tube #1 and add it to tube #2 and vortex completely. Repeat the serial dilutions for tubes #3 through #6. The concentration of Osteopontin in tubes #1 through #6 will be 20, 10, 5, 2.5, 1.25, and 0.625 ng/mL. Use all Standards within 2 hours of preparation. Stock Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Assay Buffer Volume (μL) 250 250 250 250 250 250 Addition Vial Stock Std 1 Std 2 Std 3 Std 4 Std 5 Volume of Addition (μL) 500 250 250 250 250 250 250 Final Conc (ng/ mL) 40 20 10 5 2.5 1.25 0.625

EDTA plasma samples must be diluted ≥ 1:20 with the provided Assay Buffer prior to running in the kit. This recommended dilution will allow detection of most normal plasma samples. It may be necessary to dilute high or diseases state plasmas greater than ≥ 1:100. Urine samples must be diluted ≥ 1:10 with the provided Assay Buffer prior to running in the kit. This recommended dilution will allow detection of most normal urine samples. It may be neces- sary to dilute high or diseases state urines greater than ≥ 1:40. Milk samples should be clarified prior to being run. Centrifuge the sample at 10,000 rcf for 15 min- utes at 4 °C. Using a plastic pipet tip pierce the top layer on the centrifuged sample and remove the lower supernatant. Repeat the centrifugation and supernatant isolation once more. Milk samples must be diluted with the provided Assay Buffer prior to running in the kit. A dilution of 1:2,000 or greater is recommended to detect most milk samples within the standard curve range. TCM samples should be diluted in TCM and read off a standard curve generated in the same TCM or diluted ≥1: 20 in Assay Buffer and read off a standard curve generated in Assay Buffer. Any samples with Osteopontin concentrations outside the standard curve range should be diluted further with Assay Buffer or TCM, as appropriate, to obtain readings within the standard curve range. It is up to the end user to determine the appropriate dilution for their samples. Use all samples within 2 hours of dilution.

If you believe that any of your samples may have high OPN levels in them (such as milk samples) we would recommend using the 40 ng/mL Stock OPN as an additional standard. In this case the assay must be read using the DualRead™ system by reading the plate at 650 nm prior to addition of stop solution. 1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC. 2. Pipet 50 μL of samples or standards into wells in the plate. Pipet 50 μL of Assay Buffer into the zero standard wells. 3. Incubate at room temperature for 60 minutes. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels. 4. Add 50 μL of the DetectX® Osteopontin Conjugate to each well, using a repeater pipet. 5. Incubate at room temperature for 60 minutes. 6. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels. 7. Add 100 μL of the TMB Substrate to each well, using a repeater pipet. 8. Incubate the plate at room temperature for 30 minutes. DualRead™ If the blue substrate color of any of your samples appears darker than the 20 ng/mL standard, we recommend reading the plate at 650 nm one minute prior to adding stop solution. 9. Add 50 μL of the Stop Solution to each well, using a repeater pipet. 10. Read the optical density generated from each well at 450 nm. 11. Use the plate reader's built-in 4PLC software capabilities to calculate Osteopontin concentration for each sample.

calculation of results Average the duplicate OD readings for each standard and sample. Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit. The sample concentrations should be multiplied by the dilution factor to obtain neat sample values. tyPical data Sample Mean OD Mean OD Human Osteopontin Conc. (ng/mL) (650nm) (450 nm) Alt. Standard 1.813 - 40 Standard 1 0.797 1.987 20 Standard 2 0.262 0.662 10 Standard 3 0.111 0.284 5 Standard 4 0.067 0.144 2.5 Standard 5 0.051 0.094 1.25 Standard 6 0.046 0.076 0.625 B0 0.040 0.057 0 Sample 1 0.627 1.859 17.1 (650 nm)/19.2 (450 nm) Sample 2 0.284 0.631 10.4 (650 nm)/9.53 (450 nm) Always run your own standard curve for calculation of results. Do not use this data. Optional optical density measurement at 650 nm can be performed if any samples ap- pear to generate blue color with TMB that would be in excess of the 20 ng/mL OPN standard. Typical Standard Curve # Optical Density # OD 450nm OD 650nm # # # # ! !# " =abS]^]\bW\ 1]\Q \U [: Always run your own standard curve for calculation of results. Do not use this data.