High Sensitive Bovine Non-esterified Fatty Acid ELISA Assay Kit Customized NEFA ELISA Test Kit

Cat.No E0021Bo

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 kit should not be used beyond the expiration date.

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%

Intended Use
This sandwich kit is for the accurate quantitative detection of Bovine Non-esterified Fatty Acid (also known as NEFA) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

Assay Principle
This ELISA Assay Kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Bovine NEFA antibody. NEFA present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Bovine NEFA Antibody is added and binds to NEFA in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated NEFA 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 NEFA. 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 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.

Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (640μmol/L) with 120μl of standard diluent to generate a 320μmol/L 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 (320μmol/L) 1:2 with standard diluent to produce 160μmol/L, 80μmol/L, 40μmol/L and 20μmol/L solutions. Standard diluent serves as the zero standard(0 μmol/L). 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.