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User Manual

ELK16J0215, Rabbit Osteopontin (OPN)

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Rabbit OPN ELISA Kit instruction
Intended use
This OPN ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or
therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the
color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of OPN in
the sample, this OPN ELISA Kit includes a set of calibration standards. The calibration standards are
assayed at the same time as the samples and allow the operator to produce a standard curve of Optical
Density versus OPN concentration. The concentration of OPN in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
Sample collection and storages
Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for
10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples
at -20℃or -80℃.Avoid repeated freeze-thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes
at 3000×g at 2-8℃within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated
freeze-thaw cycles.
Cell culture supernates and other biological fluids - Remove particulates by centrifugation and
assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Note: The samples shoule be centrifugated dequately and no hemolysis or granule was
allowed.
Materials required but not supplied

1. Standard microplate reader(450nm).
2. Precision pipettes and Disposable pipette tips.
3. 37 ℃incubator.
   Precautions

   6.0ml	3.0ml
   6.0ml 6.0ml	3.0ml 3.0ml
   membrane 2	2
   1 Standard (S0 ￫S5) concentration was followed by:0,10,20,40,80,160 ng/mL.	1
   20×wash solution:Dilute with Distilled or deionized water 1:20.
   Prepare all reagents before starting assay procedure. It is recommended that all Standards and
   Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
   Add Sample: Add testing sample 10μl then add 40μl of Sample Diluent to testing sample wel.
   any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper
   towels.

   4. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and
      incubate for 15 minutes at 37°C. Protect from light.
   5. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the
      color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure
      thorough mixing.
      8 Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
      Calculation of results
   6. This standard curve is used to determine the amount in an unknown sample. The standard curve is
      generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on
      the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
   7. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by
      the mean value of the zero standard before result interpretation. Construct the standard curve using graph
      paper or statistical software.
   8. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a
      horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and
      read the corresponding concentration.
   9. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age
      can cause variation in result. Each user should obtain their own standard curve.
   10. The sensitivity by this assay is 1 ng/mL.
   11. Standard curve.
       standards concentration (X)
       Storage: 2-8℃. Validity:six months.
       FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!