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|Sample:||Serum,plasma,urine,tissue,cell Culture Supernatant||Shipping:||DHL/FedEX|
sandwich elisa kit,
rat elisa kit
96 Wells High Precision and Sensitive Mouse OPN Sandwich ELISA Kit
Standard Curve Range: 0.05ng/ml - 15ng/ml
*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.
This sandwich kit is for the accurate quantitative detection of Mouse Osteopontin (also known as OPN) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Mouse OPN antibody. OPN present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Mouse OPN Antibody is added and binds to OPN in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated OPN antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Mouse OPN. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.
Serum Allow serum to clot for 10-20 minutes at room temperature. Centrifuge at 2000-3000 RPM for 20 minutes.
Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C within 30 minutes of collection.
Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for approximately 20 minutes. When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow the procedures above-mentioned.
Cell Culture Supernatant Collect by sterile tubes when examining secrete components. Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for approximately 20 minutes.
Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20 minutes.
*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample matrix interference may falsely depress the specificity and accuracy of the assay.
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (16ng/ml) with 120μl of standard diluent to generate a 8ng/ml standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (8ng/ml) 1:2 with standard diluent to produce 4ng/ml, 2ng/ml, 1ng/ml and 0.5ng/ml solutions. Standard diluent serves as the zero standard(0 ng/ml). Any remaining solution should be frozen at -20℃ and used within one month. Dilution of standard solutions suggested are as follows:
|8ng/ml||Standard No.5||120μl Original Standard + 120μl Standard Diluent|
|4ng/ml||Standard No.4||120μl Standard No.5 + 120μl Standard Diluent|
|2ng/ml||Standard No.3||120μl Standard No.4 + 120μl Standard Diluent|
|1ng/ml||Standard No.2||120μl Standard No.3 + 120μl Standard Diluent|
|0.5ng/ml||Standard No.1||120μl Standard No.2 + 120μl Standard Diluen|
|Standard Concentration||Standard No.5||Standard No.4||Standard No.3||Standard No.2||Standard No.1|
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 500 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.
1. Prepare all reagents, standard solutions and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature.
2. Determine the number of strips required for the assay. Insert the strips in the frames for use. The unused strips should be stored at 2-8°C.
3. Add 50μl standard to standard well. Note: Don’t add antibody to standard well because the standard solution contains biotinylated antibody.
4. Add 40μl sample to sample wells and then add 10μl anti-OPN antibody to sample wells, then add 50μl streptavidin-HRP to sample wells and standard wells (Not blank control well). Mix well. Cover the plate with a sealer. Incubate 60 minutes at 37°C.
5. Remove the sealer and wash the plate 5 times with wash buffer. Soak wells with at least 0.35 ml wash buffer for 30 seconds to 1 minute for each wash. For automated washing, aspirate all wells and wash 5 times with wash buffer, overfilling wells with wash buffer. Blot the plate onto paper towels or other absorbent material.
6. Add 50μl substrate solution A to each well and then add 50μl substrate solution B to each well. Incubate plate covered with a new sealer for 10 minutes at 37°C in the dark.
7. Add 50μl Stop Solution to each well, the blue color will change into yellow immediately.
8. Determine the optical density (OD value) of each well immediately using a microplate reader set to 450 nm within 10 minuets after adding the stop solution.
Calculation of Result
Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.
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