β-Hydroxybutyrate (Ketone Body) Colorimetric Assay Kit

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β-Hydroxybutyrate (Ketone Body) Colorimetric Assay Kit

SKU #	E-BC-K785-M
Detection Instrument	Microplate reader (440-460 nm, optimum wavelength: 450 nm)
Detection method:	Colorimetric method

Product Details

Properties

Synonyms	β-HB
Sample type	Serum, plasma, urine, animal tissue
Sensitivity	0.01 mmol/L
Detection range	0.01-2.00 mmol/L
Detection method	Colorimetric method
Assay type	Quantitative
Assay time	60 min
Precision	Average inter-assay CV: 4% \| Average intra-assay CV: 2%
Other instruments required	Incubator, 50 kD Ultrafiltration tube, Microplate reader
Storage	2-8℃
Valid period	12 months

Dilution of Sample

The recommended dilution factor for different samples is as follows (for reference only):

Sample type	Dilution factor
Human serum	1
Human plasma	1
Rat /Mouse serum	1
Rat /Mouse plasma	1
10% Rat liver tissue homogenate	1
10% Rat kidney tissue homogenate	1
10% Rat heart tissue homogenate	1
Human urine	1

Note: The diluent is double distilled water. For the dilution of other sample types, please do pretest to confirm the dilution factor.

Detection Principle

β- hydroxybutyrate dehydrogenase can catalyze the oxidative dehydrogenation of β-HB. Meanwhile, NAD+ is reduced to NADH, Which under the action of hydrogen transmitter, transfer electrons to WST-8 to produce the yellow product. The content of β-HB can be calculated by measuring the change of absorbance value at 450 nm.

Kit Components & Storage

Item	Component	Size 1(48 T)	Size 2(96 T)	Storage
Reagent 1	Buffer Solution A	50 mL × 1 vial	50 mL × 2 vials	2-8℃, 12 months, shading light
Reagent 2	Enzyme Reagent	Powder × 1 vial	Powder × 2 vials	2-8℃, 12 months, shading light
Reagent 3	Buffer Solution B	5 mL × 1 vial	10 mL × 1 vial	2-8℃, 12 months
Reagent 4	Chromogenic Agent	1.5 mL× 1 vial	1.5 mL × 2 vials	2-8℃, 12 months, shading light
Reagent 5	10 mmol/L Standard	0.5 mL × 1 vial	1 mL × 1 vial	2-8℃, 12 months
	Microplate	96 wells		No requirement
	Plate Sealer	2 pieces

Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.

Technical Data:

Parameter:

Intra-assay Precision

Three human serum samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).

Parameters	Sample 1	Sample 2	Sample 3
Mean (mmol/L)	0.55	1.05	1.50
%CV	2.3	2.0	1.7

Inter-assay Precision

Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.

Parameters	Sample 1	Sample 2	Sample 3
Mean (mmol/L)	0.55	1.05	1.50
%CV	3.8	4.2	4.0

Recovery

Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 105%.

	Standard 1	Standard 2	Standard 3
Expected Conc. (mmol/L)	0.25	0.85	1.3
Observed Conc. (mmol/L)	0.3	0.9	1.4
Recovery rate(%)	104	106	105

Sensitivity

The analytical sensitivity of the assay is 0.01 mmol/L. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.

Standard Curve

As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:

Concentration (mmol/L)	0	0.2	0.5	0.8	1.0	1.2	1.5	2.0
Average OD	0.061	0.139	0.254	0.390	0.463	0.540	0.667	0.879
Absoluted OD	0	0.078	0.193	0.329	0.402	0.479	0.606	0.818