NOT FOR USE IN DIAGNOSTIC PROCEDURES.

CanineThyroid Stimulating Hormone(TSH)ELISA Kit instruction

Intended use

This TSH 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 TSH in the sample, this TSH 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 TSH concentration. The concentration of TSH in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Samplecollection 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 30minutes 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

1. Do not substitute reagents from one kit to another. Standard, conjugate ａnd microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

3. Mix all reagents before using.

Remove all kit reagents from refrigerator ａnd allow them to reach room temperature ( 20-25°C)

Note: Standard concentration was followed by:

300、150、75、37.5、18.75、9.375pg/mL.

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1. Prepare all r e a ge nt s before starting assay procedure. It is recommended that all Standards ａnd Samples be added in duplicate to the Microelisa Stripplate.

2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.

3. Add Sample: Add testing sample10μl Then add sample diluent40μl to testing sample well; Blank welldoesn’t add anyting.

4. Add 100μl of HRP-conjugate reagentto each well,cover with anadhesive stripand incubate for 60 minutes at 37°C.

5. Aspirate each well ａnd wash, repeating the process fourtimes for a total of five washes.Wash by filling each well with Wash Solution(400μl) using a squirt bottle, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solutionby aspirating ordecanting. Invert the plate ａnd blot it against clean paper towels.

6. Add chromogen solution A 50μl ａnd chromogen solution B 50μl to each well.Gently mix ａnd incubate for 15 minutes at 37°C. Protectfrom light.

7. Add 50μl Stop Solution to each well. The color in the wells should change from blue toyellow. 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

1. 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.

2. 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.

3. 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.

4. 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.

5. The sensitivity by this assay is1.0pg/mL.

6. 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!