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  1. Top
  2. Life Science
  3. Metabolism Research
  4. ELISA / Assay Kits (Metabolism)
  5. Non-Esterified Fatty Acid (NEFA) Assay Kits
  6. LabAssay (TM) NEFA (FFA)

LabAssay (TM) NEFA (FFA)

for Cellbiology
Manufacturer :
FUJIFILM Wako Pure Chemical Corporation
Storage Condition :
Keep at 2-10 degrees C.
GHS :
Citations (4)
PubMed Central® search
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SDS
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Product Number
Package Size
Price
Inventory
Distributor
299-94301
Barcode No
4548995103444
500Tests
List Price
JPY 49,000

In stock in Japan

Please check here for notes on products and prices.

Document

SDS
Product Specification Sheet
Package Insert
Spectral Data
Certificate of Analysis
Calibration Certificate
Analytical Charts

Kit component

For 500 tests

Chromogen Reagent A for 10 mL x 4 bottles
Solvent A (for Chromogen Reagent A) 45 mL x 1 bottle
Chromogen Reagent B for 20 mL x 4 bottles
Solvent B (for Chromogen Reagent B) 90 mL x 1 bottle
Standard Solution 7 mL x 1 bottle

Product Overview

NEFA (Non‐esterified fatty acid) in the blood is transported complexed with an albumin to peripheral tissues. They are important sources of fuel for the peripheral tissues. The concentration of NEFA in the blood is regulated by a release from the adipose tissues, a consumption in the periphral tissues or a take up from the liver.

LabAssay™ NEFA (FFA) is a kit designed to measure non-esterified fatty acids (NEFA) in samples using the ACS-ACOD method.

[Note] LabAssay™ series are reagents for research purposes. They cannot be used for diagnostic purposes.

Kit Performance

Analysis sample Human Serum/Plasma
Mouse Serum/Plasma
Rat Serum/Plasma
Dog Serum/Plasma
Cat Serum/Plasma
Culture medium (D-MEM)*
Calibration curve range 0.4-1.97 mEq/L
*Oleic acid 1 mEq=1 mmol
Sample volume 4 μL
Measurement duration Approx. 20 min
Wavelength 550 nm
*It is tested by using the samples that standard substances have been added to the culture medium.
Measurement availability depends on the culture medium, cell type and culture conditions. Please perform a spiked recovery test or dilution linearity test in advance using the culture medium to be used in your experiment.

Example of Calibration Curve

Example of Calibration Curve

Assay Principle

NEFA (Non‐esterified fatty acid) forms Acyl-CoA in the presence of Acyl-CoA synthetase (ACS). Hydrogen peroxide, which is produced by a reaction between the Acyl-CoA and Acyl-CoA oxidase (ACOD), promotes oxidative condensation of 3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline (MEHA) with 4-aminoantipyrine. LabAssay™ NEFA can be used for the quantitative determination of NEFA in samples by measuring absorbance of a blue purple color which is generated by the oxidative condensation reaction.

Principle of the NEFA assay using the ACS-ACOD method

Procedure

Assay in a microplate
Perform the assay in the wells according to the following table scheme.

Test Standard Reagent Blank Sample Blank *1
Color Reagent A 80 μL 80 μL 80 μL 80 μL
Specimen Serum
4 μL		 Standard
4 μL		 Distilled or
Deionized water
4 μL		 -
Mix well *2 and incubate at 37°C for 10 min.
Color Reagent B 160 μL 160 μL 160 μL 160 μL
Specimen - - - Serum
4 μL
Mix well *2 and incubate at 37℃ for 10 min.
After cooling the reaction solution to room temperature, measure the absorbance at 550 nm of the test sample and standard solution with the blank solution as the control within the next 30 min.

*1 Sample blank is not required for common samples, but required for chyle or hemolyzed sample.
*2 Mix well with a plate mixer at 600 - 1,000 rpm for 1 minute. Be careful not to spill from the well

Data

Repeatability (within run precision)

Repeatability was conducted by measuring sixfold assays of serum/plasma (EDTA) samples from human, mouse and rat at two concentrations with this product.

n＼ID Human serum Mouse serum Rat serum
ID1 (mEq/L) ID2 (mEq/L) ID1 (mEq/L) ID2 (mEq/L) ID1 (mEq/L) ID2 (mEq/L)
1 0.872 0.540 1.439 0.523 0.936 0.542
2 0.893 0.538 1.483 0.505 0.933 0.565
3 0.891 0.554 1.499 0.522 0.941 0.566
4 0.890 0.551 1.449 0.543 0.948 0.561
5 0.928 0.595 1.439 0.541 0.931 0.547
6 0.922 0.608 1.470 0.518 0.928 0.526
mean 0.899 0.564 1.463 0.525 0.936 0.551
SD 0.0214 0.0297 0.02487 0.0144 0.00731 0.0158
CV(%) 2.4 5.3 1.7 2.7 0.8 2.9

[Result]
The CV (%) of human serum was 2.4-5.3%, mouse serum was 1.7-2.7% and rat serum was 0.8-2.9%, indicating good repeatability.

Reproducibility (between run precision)

Reproducibility was conducted by measuring serum samples from human, mouse and rat at three concentrations each for four days with this product.

Day＼ID Human serum Mouse serum Rat serum
ID3 (mEq/L) ID4 (mEq/L) ID5 (mEq/L) ID3 (mEq/L) ID4 (mEq/L) ID5 (mEq/L) ID3 (mEq/L) ID4 (mEq/L) ID5 (mEq/L)
1 0.899 0.630 0.486 1.018 0.802 0.596 0.726 0.562 0.434
2 0.837 0.591 0.449 1.019 0.784 0.582 0.836 0.632 0.477
3 0.910 0.645 0.491 1.021 0.787 0.580 0.800 0.612 0.465
4 0.923 0.649 0.487 1.020 0.791 0.585 0.824 0.620 0.479
mean 0.892 0.629 0.478 1.020 0.791 0.586 0.797 0.607 0.464
SD 0.0381 0.0265 0.0196 0.001291 0.00787 0.00714 0.0493 0.0308 0.0208
CV(%) 4.3 4.2 4.1 0.1 1.0 1.2 6.2 5.1 4.5

[Result]
The CV (%) of human serum was 4.1-4.3%, mouse serum was 0.1-1.2% and rat serum was 4.5-6.2%, indicating good reproducibility.

Dilution Linearity Test

Serum and plasma (EDTA) from human, mouse and rat, as well as D-MEM containing 10% FBS spiked with the standard, were serially diluted twofold with saline and measured in duplicate to assess linearity.

human serum
mouse serum
rat serum
D-MEM/10% FBS medium

[Result]
All measurement results showed good linearity within the measurement range.

Spike and Recovery Test

Standard solutions at four concentrations were added to serum/plasma (EDTA) from human, mouse and rat. Next, spike recovery tests were conducted, with each measurement performed in duplicate.

Human sample

Spiked amount
(mEq/L)		 Measurement value
(mEq/L)		 Recovery volume
(mEq/L)		 Recovery rate
(%)
Serum - 0.69 - -
0.40 1.14 0.45 113
0.61 1.34 0.64 105
0.78 1.54 0.85 109
1.01 1.73 1.03 102
Average 107
Plasma
(EDTA)		 - 0.28 - -
0.40 0.70 0.43 108
0.61 0.90 0.62 102
0.78 1.12 0.84 108
1.01 1.34 1.06 105
Average 106

Mouse sample

Spiked amount
(mEq/L)		 Measurement value
(mEq/L)		 Recovery volume
(mEq/L)		 Recovery rate
(%)
Serum - 0.70 - -
0.40 1.07 0.37 92.5
0.61 1.31 0.61 100
0.78 1.44 0.74 94.9
1.01 1.62 0.92 91.1
Average 94.6
Plasma
(EDTA)		 - 0.62 - -
0.40 0.99 0.37 92.5
0.61 1.29 0.68 111
0.78 1.38 0.77 98.7
1.01 1.62 1.00 99.0
Average 101

Rat sample

Spiked amount
(mEq/L)		 Measurement value
(mEq/L)		 Recovery volume
(mEq/L)		 Recovery rate
(%)
Serum - 0.57 - -
0.40 1.00 0.43 108
0.61 1.21 0.65 107
0.78 1.40 0.83 106
1.01 1.57 1.01 100
Average 105
Plasma
(EDTA)		 - 0.41 - -
0.40 0.86 0.45 113
0.61 1.07 0.66 108
0.78 1.27 0.86 110
1.01 1.44 1.02 101
Average 110

[Result]
Good recovery rates were confirmed.

Measurement Using Actual Samples

Human sample

ID＼n Measurement value
(mEq/L)		 mean SD CV
(%)
1 2
Serum ID1 0.686 0.688 0.687 0.00141 0.2
Plasma (EDTA) ID1 0.354 0.350 0.352 0.00283 0.8

Rat sample (S.D. rat)

ID＼n Measurement value
(mEq/L)		 mean SD CV
(%)
1 2
Seum ID1 0.300 0.300 0.300 0.00 0.0
ID2 0.373 0.368 0.371 0.00354 1.0
ID3 0.301 0.277 0.289 0.0170 5.9
ID4 0.365 0.364 0.365 0.000707 0.2
ID5 0.403 0.362 0.383 0.0290 7.6
Plasma
(EDTA)		 ID1 0.336 0.330 0.333 0.00377 1.1
ID2 0.358 0.354 0.356 0.00283 0.8
ID3 0.311 0.339 0.325 0.0196 6.0
ID4 0.222 0.214 0.218 0.00566 2.6
ID5 0.522 0.508 0.515 0.00990 1.9

Mouse sample (CD-1(ICR))

ID＼n Measurement value
(mEq/L)		 mean SD CV
(%)
1 2
Seum ID1 1.119 1.117 1.118 0.001414 0.1
ID2 0.783 0.793 0.788 0.00707 0.9
ID3 0.700 0.731 0.716 0.0219 3.1
ID4 0.795 0.792 0.794 0.00212 0.3
ID5 0.825 0.833 0.829 0.00566 0.7
Plasma
(EDTA)		 ID1 0.311 0.308 0.310 0.00212 0.7
ID2 0.319 0.321 0.320 0.00141 0.4
ID3 0.202 0.208 0.205 0.00424 2.1
ID4 0.173 0.185 0.179 0.00849 4.7
ID5 0.293 0.318 0.306 0.0177 5.8

Dog sample (Beagle)

ID＼n Measurement value
(mEq/L)		 mean SD CV
(%)
1 2
Serum ID1 0.654 0.657 0.656 0.00212 0.3
ID2 0.642 0.688 0.665 0.0325 4.9
ID3 0.419 0.431 0.425 0.00849 2.0
ID4 0.377 0.383 0.380 0.00424 1.1
ID5 0.887 0.877 0.882 0.00707 0.8
ID6 0.604 0.595 0.600 0.00636 1.1
ID7 0.987 1.025 1.006 0.02687 2.7
ID8 0.501 0.510 0.506 0.00636 1.3
Plasma
(EDTA)		 ID1 0.440 0.444 0.442 0.00283 0.6
ID2 0.419 0.435 0.427 0.0113 2.6
ID3 0.311 0.306 0.309 0.00354 1.1
ID4 0.470 0.487 0.479 0.0120 2.5
ID5 0.472 0.475 0.474 0.00212 0.4
ID6 0.370 0.364 0.367 0.00424 1.2
ID7 0.789 0.770 0.780 0.0134 1.7
ID8 0.439 0.384 0.412 0.0389 9.5

Cat sample (Short hair)

ID＼n Measurement value
(mEq/L)		 mean SD CV
(%)
1 2
Serum ID1 0.709 0.726 0.718 0.0120 1.7
ID2 0.628 0.642 0.635 0.00990 1.6
ID3 1.004 1.003 1.004 0.007071 0.1
ID4 0.974 0.987 0.981 0.00919 0.9
ID5 0.624 0.649 0.637 0.0177 2.8
ID6 1.175 1.212 1.194 0.02616 2.2
ID7 1.086 0.959 1.023 0.08980 8.8
ID8 0.371 0.393 0.382 0.0156 4.1
Plasma
(EDTA)		 ID1 1.092 1.107 1.100 0.01061 1.0
ID2 0.177 0.172 0.175 0.00354 2.0
ID3 0.243 0.240 0.242 0.00212 0.9
ID4 0.320 0.319 0.320 0.000707 0.2
ID5 0.363 0.354 0.359 0.00636 1.8
ID6 0.206 0.189 0.198 0.0120 6.1
ID7 0.232 0.199 0.216 0.0233 10.8
ID8 0.142 0.120 0.131 0.0156 11.9

FAQ

About sample

Is there any influence from substances that interfere with measurement (ascorbic acid, bilirubin, etc.)?
Bilirubin may cause a negative bias, whereas ascorbic acid has little to no effect on the assay results.

About kit usage

What instruments, and equipment are required for the assay using this kit?
The instruments and equipment required for the use of this kit are listed below.
  • 96 well microplate (transparent type)
  • Micropipette
  • Plate mixer
  • Incubator maintaining at 37°C
  • Microplate reader with 550 nm wavelength filter

(For test tube method)

  • Test tube or microtube
  • Pipette
  • Incubator maintaining at 37°C
  • Spectrophotometer or colorimeter with a 550 nm wavelength filter
What are the storage conditions and shelf-life guidelines for each reagent?

Color Reagent A :
Prepare Reagent A by mixing 1 vial of each Chromogen Reagent A (for 10 mL) and Solvent A (for Chromogen Reagent A) (10 mL). After reconstitution, the solution should be stored below 25°C and used the same day, or stored at 2°C - 10°C and used within 5 days.

Color Reagent B:
Prepare Reagent B by mixing 1 vial of each Chromogen Reagent B (for 20 mL) and Solvent B (for Chromogen Reagent B) (20 mL). After reconstitution, the solution should be stored below 25°C and used the same day, or stored at 2°C - 10°C and used within 5 days.

Overview / Applications

Property

Manufacturer Information

Alias

  • LabAssay
    633-52001 (Previous Code)
    LABNEFA-M1 (Previous Code)
    294-63601 (Previous Code)

For research use or further manufacturing use only. Not for use in diagnostic procedures.

Product content may differ from the actual image due to minor specification changes etc.

If the revision of product standards and packaging standards has been made, there is a case where the actual product specifications and images are different.

The prices are list prices in Japan.Please contact your local distributor for your retail price in your region.

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