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User Manual

ELK11J0054, Canine Leptin (LEP)

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standards concentration (X)
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Canine LEP ELISA Kit instruction
Intended use
This LEP 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 LEP in the sample, this LEP 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 LEP concentration. The concentration of LEP 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

   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 0.1 ng/mL.
   11. Standard curve.
       Storage: 2-8℃. Validity:six months.
       FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE
       BEGINNING!