Creatinine (Cr) Content Assay Kit (Creatine Oxidase, Micro Method)

CAS: 3047612-66-5 Cat. No.: C1525442

GRADE & PURITY BioReagent, Suitable for Analysis, Colorimetry

Overview

Creatinine is converted to creatine under the catalysis of creatinine amidohydrolase, and creatine is further converted to sarcosine and urea by creatine amidohydrolase. Sarcosine is then oxidized by sarcosine oxidase to produce glycine, formaldehyde, and hydrogen peroxide. Hydrogen peroxide reacts with a chromogenic reagent in the presence of peroxidase to form a pink compound. The change in optical density (OD) at 515 nm can be measured to determine and calculate the creatinine concentration.  

Kit Contents

C1525442	Components	Appearance	48T	96T	Storage	Quantity Per Test
C1525442A	Reagent 1	Yellow liquid	10 mL	20 mL	2-8℃. Store in the dark	180 uL for 1 reaction
C1525442B	Reagent 2	Pink liquid	3.5 mL	7 mL	2-8℃. Store in the dark	60 uL for 1 reaction
C1525442C	1 mM Standard Sample	Colorless  liquid	1.5 mL	1.5 mL×2	2-8℃. Store in the dark	As needed

Experimental Instruments:

Mortar/homogenizer, analytical balance, ice box/ice maker, tabletop centrifuge, adjustable pipette, water bath incubator/drying oven/cell incubator/metal bath, 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water is acceptable).

Reagent Preparation:

1. Before testing, the reagents in the kit should be equilibrated to room temperature.

2. Dilution of different concentration standards:

Number	①	②	③	④	⑤	⑥	⑦	⑧
Standard concentration (mmol/L)	0	0.1	0.15	0.2	0.25	0.3	0.35	0.4
1 mM Standard Sample (μL)	0	20	30	40	50	60	70	80
Double-distilled water (μL)	200	180	170	160	150	140	130	120

Sample Preparation:

Serum and plasma liquid samples: Direct determination. Sample dilution: Before the formal test, select 2-3 samples with expected significant differences and dilute them into different concentrations for a preliminary experiment. Based on the results of the preliminary experiment and in combination with the linear range of this kit: 0 - 400 μmol/L, you can refer to the following table for dilution (for reference only):

Sample	Dilution factor	Sample	Dilution factor
Human urine	40-60	Rat serum	No dilution
Human serum	No dilution	Pig serum	No dilution
Mouse serum	No dilution

Note: The diluent is physiological saline (0.9% NaCl).

Key points of the experiment:

1. When adding the sample to the wells of the plate, it should be added to the bottom.

2. If the sample quantity is large, it is recommended to use a pipette for dispensing. If there is no pipette, it is suggested to limit the number of wells to no more than 20.

Operation steps:

1. Standard wells: Take 12 μL of samples of different concentrations and add them to the corresponding standard wells; Sample wells: Take 12 μL of the sample to be tested and add it to the sample wells.

2. Add 180 μL of reagent one to each well in 1.

3. Incubate at 37 °C for 5 minutes, and measure the OD value of each well at 515 nm using an enzyme detector, which is A1. 4. Add 60 μL of reagent two to each of the standard and test wells. 5. Incubate at 37 °C for 5 minutes, and measure the OD value of each well at 515 nm, which is  A2.

Standard well	Measurement well
Different concentrations of standard substances (μL)	12	--
Sample (μL)	--	12
Reagent 1 (μL)	180	180
Mix, incubate at 37 ℃ for 5 minutes, read the absorbance value A1 at 515 nm.
Reagent 2 (μL)	60	60
Mix, incubate at 37 ℃ for 5 minutes, read the absorbance value A2 at 515 nm. ΔA = A₂ - A₁.

Result calculation:

Standard curve: y = ax + b

Formula for calculating creatinine content in serum (plasma) and urine: Cr content (μmol/L) = (ΔA515 - b) ÷ a × 1000 × f

Notes:

y: ΔA of the standard sample minus ΔA of the blank (ΔA at the standard sample concentration of 0)

x: Concentration of the standard sample

a: Slope of the calibration curve

b: Intercept of the calibration curve

ΔA515: Sample ΔA minus blank ΔA

*: The concentration unit in the calibration curve is mmol/L, convert it to μmol/L

f: Dilution factor of the sample before adding to the detection system

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice,Do not freeze

Stability And Storage

Each component has a shelf life of 3 months under corresponding storage conditions.

Contents & Storage

C1525442	Components	Appearance	48T	96T	Storage	Quantity Per Test
C1525442A	Reagent 1	Yellow liquid	10 mL	20 mL	2-8℃. Store in the dark	180 uL for 1 reaction
C1525442B	Reagent 2	Pink liquid	3.5 mL	7 mL	2-8℃. Store in the dark	60 uL for 1 reaction
C1525442C	1 mM Standard Sample	Colorless clear liquid	1.5 mL	1.5 mL×2	2-8℃. Store in the dark	As needed

Images

Standard Curve assay: Creatinine (Cr) Content Assay Kit (Creatine Oxidase, Micro Method) (C1525442)

Creatinine (Cr) Content Assay Kit (Creatine Oxidase, Micro Method) (C1525442) 

Creatinine is converted to creatine under the catalysis of creatinine amidohydrolase, and creatine is further converted to sarcosine and urea by creatine amidohydrolase. Sarcosine is then oxidized by sarcosine oxidase to produce glycine, formaldehyde, and hydrogen peroxide. Hydrogen peroxide reacts with a chromogenic reagent in the presence of peroxidase to form a pink compound. The change in optical density (OD) at 515 nm can be measured to determine and calculate the creatinine concentration. This detection method can measure the concentration of creatinine and shows a good linear relationship within the range of 0 - 0.4 mmol/L.