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:
320μmol/L | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
160μmol/L | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
80μmol/L | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
40μmol/L | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
20μmol/L | Standard No.1 | 120μl Standard No.2 + 120μl Standard Diluent |
Standard Concentration | Standard No.5 | Standard No.4 | Standard No.3 | Standard No.2 | Standard No.1 |
640μmol/L | 320μmol/L | 160μmol/L | 80μmol/L | 40μmol/L | 20μmol/L |
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.