Laboratory Research Bovine High Precision and Sensitivity BHBA ELISA Assay Kit 96 Well Plate

Cat.No E0267Bo

Intended Use

This sandwich kit is for the accurate quantitative detection of Bovine Beta-hydroxybutyric Acid (also known as BHBA) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

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 Bovine BHBA antibody. BHBA present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Bovine BHBA Antibody is added and binds to BHBA in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated BHBA 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 Bovine BHBA. 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 ELISA Assay 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 ELISA Assay Kit should not be used beyond the expiration date.

Reagent Preparation

All reagents should be brought to room temperature before use.

Standard Reconstitute the 120μl of the standard (4800nmol/ml) with 120μl of standard diluent to generate a 2400nmol/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 (2400nmol/ml) 1:2 with standard diluent to produce 1200nmol/ml, 600nmol/ml, 300nmol/ml and 150nmol/ml solutions. Standard diluent serves as the zero standard(0 nmol/ml). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:

Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 600 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.