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15-3 ELISA Assay Kit Laboratory Research Canine Carbohydrate Antigen With Strong Sensitivity

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15-3 ELISA Assay Kit Laboratory Research Canine Carbohydrate Antigen With Strong Sensitivity

Brand Name : BT Lab

Model Number : Cat.No E0156Ca

Certification : CE, ISO9001:2005, MSDS

Place of Origin : Shanghai, China

MOQ : Negotiation

Price : Negotiation

Supply Ability : Western Union, T/T

Delivery Time : 1-3 business days, bulk order within one week

Packaging Details : Wrapped with ice pack and styrofoam package

Known as : CA15-3

OEM : Acceptable

In Stock : Yes

Shipping : DHL/FedEX

Discount : Available

Organism Species : Canine

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Laboratory Research Canine Carbohydrate Antigen 15-3 ELISA Assay Kit With Strong Sensitivity


Cat.No E0156Ca


Assay Procedure

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-CA15-3 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.


Standard Curve Range: 0.2U/ml - 60U/ml

Sensitivity: 0.096U/ml

Size: 96 wells

Storage: Store the reagents at 2-8°C. For over 6-month storage refer to the expiration date keep it at -20°C. Avoid repeated thaw cycles. If individual reagents are opened it is recommended that the kit be used within 1 month.

*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%


Assay Principle

This elisa assay kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Canine CA15-3 antibody. CA15-3 present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Canine CA15-3 Antibody is added and binds to CA15-3 in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated CA15-3 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 Canine CA15-3. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.


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 and 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.


Reagent Preparation


All reagents should be brought to room temperature before use.

Standard Reconstitute the 120μl of the standard (64U/ml) with 120μl of standard diluent to generate a 32U/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 (32U/ml) 1:2 with standard diluent to produce 16U/ml, 8U/ml, 4U/ml and 2U/ml solutions. Standard diluent serves as the zero standard(0 U/ml). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:


32U/mlStandard No.5120μl Original Standard + 120μl Standard Diluent
16U/mlStandard No.4120μl Standard No.5 + 120μl Standard Diluent
8U/mlStandard No.3120μl Standard No.4 + 120μl Standard Diluent
4U/mlStandard No.2120μl Standard No.3 + 120μl Standard Diluent
2U/mlStandard No.1120μl Standard No.2 + 120μl Standard Diluent

Standard ConcentrationStandard No.5Standard No.4Standard No.3Standard No.2Standard No.1
64U/ml32U/ml16U/ml8U/ml4U/ml2U/ml

Wash Buffer Dilute 20ml of Wash Buffer Concentrate 25x 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.


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.


Product Tags:

elisa reagent kit

      

elisa kit

      
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