This kit is based on Competitive-ELISA detection method and takes 2h assay time. The microplate provided in this kit has been precoated with FFA. FFA in the sample or standard competes with a fixed amount of biotin-labeled FFA for sites on a pre-coated antibody specific to FFA. Free components are washed off. After adding HRP-Streptavidin Conjugate (SABC), biotin specifically binds with streptavidin to form immune complexes. Wash off unbound conjugate. Add TMB substrate solution. Then, TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. Compare OD450 value of sample with the standard curve through curve fitting software to determine the concentration of FFA in the sample. The concentration of the target substance is inversely proportional to the OD450 value.
Specification
| Product Name | Bovine FFA (free fatty acid) ELISA Kit |
| Alias | NEFA ELISA Kit, FFA ELISA Kit, free fatty acid ELISA Kit, non-esterified fatty acid ELISA Kit |
| Sample Type | Serum, Plasma, Cell Culture Supernatant, cell or tissue lysate, Other liquid samples |
| Detection Method | Competitive ELISA, Coated with Antigen |
| Detection Wavelength | OD450 |
| Reaction Duration | 2 hours |
| Storage | 2-8°C(Sealed), Don't cryopreserve. |
| Specificity | Specifically binds with FFA , no obvious cross reaction with other analogues. |
Standard Curve
This product is detected by QC department and meets performance required in the manual. (Laboratory Humidity: 20%-60%; Temperature: 18°C -25°C; Equilibrate TMB substrate to 37°C before staining. After adding into the ELISA wells, incubate for 15min at 37°C in dark.). Due to different assay environments and operations, assay data below and standard curve are provided for reference. Experimenters should establish standard curve according to their own assay.
| STD.(ng/ml) | OD-1 | OD-2 | Average |
| 0 | 2.498 | 2.57 | 2.534 |
| 6.25 | 1.684 | 1.732 | 1.708 |
| 12.5 | 1.119 | 1.151 | 1.135 |
| 25 | 0.777 | 0.799 | 0.788 |
| 50 | 0.454 | 0.468 | 0.461 |
| 100 | 0.292 | 0.3 | 0.296 |
| 200 | 0.185 | 0.191 | 0.188 |
| 400 | 0.119 | 0.123 | 0.121 |
Recovery
Add a certain amount of FFA into the sample. Calculate the recovery by comparing the measured value with the expected amount of FFA in the sample.
| Sample Type | Recovery Range(%) | Average(%) |
| serum(n=10) | 95-103 | 100 |
| EDTA plasma(n=10) | 87-105 | 92 |
| Heparin plasma(n=10) | 87-93 | 90 |
Linearity
Dilute the sample with a certain amount of FFA at 1:2, 1:4 and 1:8 to get the recovery range.
| Sample Type | 1:2 | 1:4 | 1:8 |
| serum(n=10) | 85-104% | 92-100% | 87-96% |
| EDTA plasma(n=10) | 83-100% | 84-99% | 82-96% |
| Heparin plasma(n=10) | 85-94% | 80-100% | 83-97% |
Precision(%)
Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.
Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.
| Item | Intra-assay Precision | Inter-assay Precision | ||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (nmol/ml) | 12.84 | 48.8 | 201.7 | 12.78 | 50.23 | 206.1 |
| Standard deviation | 0.68 | 2.24 | 9.36 | 0.6 | 2.3 | 9.69 |
| CV(%) | 5.3 | 4.59 | 4.64 | 4.67 | 4.57 | 4.7 |
Stability
Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.
| ELISA kit(n=5) | 37°C for 1 month | 2-8°C for 6 months |
| Average(%) | 80 | 95-100 |
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