96 Wells Mouse ELISA Kit 2-8°C Storage For Accurate Quantitative Detection

96 Wells Mouse ucOC ELISA Kit High Specificity For Accurate Quantitative Detection

Cat.No EE1209Mo

*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.

Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.

CV(%) = SD/mean x 100

Intra-Assay: CV<8%

Inter-Assay: CV<10%

Intended Use

This sandwich kit is for the accurate quantitative detection of Mouse Undercarboxylated Osteocalcin (also known as ucOC) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

Assay Principle

This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Mouse ucOC antibody. ucOC present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Mouse ucOC Antibody is added and binds to ucOC in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated ucOC 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 ucOC. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.

Reagent Provided

Material Required But Not Supplied

• 37°C±0.5°C incubator
• Absorbent paper
• Precision pipettes and disposable pipette tips
• Clean tubes
• Deionized or distilled water
• Microplate reader with 450 ± 10nm wavelength filter

Precautions

• Prior to use, the kit and sample should be warmed naturally to room temperature 30 minutes.
• This instruction must be strictly followed in the experiment.
• Once the desired number of strips has been removed, immediately reseal the bag to protect the remain from deterioration. Cover all reagents when not in use.
• Make sure pipetting order and rate of addition from well-to-well when pipetting reagents.
• Pipette tips and plate sealer in hand should be clean and disposable to avoid cross-contamination.
• Avoid using the reagents from different batches together.
• Substrate solution B is sensitive to light, don’t expose substrate solution B to light for a long time.
• Stop solution contains acid. Please wear eye, hand and skin protection when using this material. Avoid contact of skin or mucous membranes with kit reagent.
• The kit should not be used beyond the expiration date.

Specimen Collection

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 other body fluids 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.

Note

• Sample concentrations should be predicted before being used in the assay. If the sample concentration is not within the range of the standard curve, users must contact us to determine the optimal sample for their particular experiments.
• Samples to be used within 5 days should be stored at 2-8°C. Samples should be aliquoted or must be stored at -20°C within 1 month or -80°C within 6 months. Avoid repeated freeze thaw cycles.
• Samples should be brought to room temperature before starting the assay.
• Centrifuge to collect sample before use.
• Samples containing NaN3 can’t be tested as it inhibits the activity of Horse Radish Peroxidase (HRP).
• Collect the supernatants carefully. When sediments occurred during storage, centrifugation should be performed again.
• Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.

*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.

Summary

1. Prepare all reagents, samples and standards.

2. Add sample and ELISA reagent into each well. Incubate for 1 hour at 37°C.

3. Wash the plate 5 times.

4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.

5. Add stop solution and color develops.

6. Read the OD value within 10 minutes.

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.