Creatinine (Cr) Content Assay Kit (PA Rate, Micro Method)

Cat. No.: C1505879

GRADE & PURITY BioReagent

Overview

Creatinine (Cr) is a metabolic product of human or animal muscle. Approximately 1 mg of creatinine is produced for every 20 g of muscle metabolized. It is filtered and excreted by the glomeruli. Exogenous creatinine originates from the metabolism of meat in the diet, while endogenous creatinine is derived from the metabolism of muscle tissue within the body.

Assay Principle

This assay measures the reaction rate between creatinine in serum, plasma, or urine and picrate, forming an orange-red creatinine-picrate complex. By selecting an appropriate rate monitoring time, interference from substances that affect the creatinine-picrate reaction is minimized. The absorbance at 510 nm is measured using a microplate reader. This kit can be used to determine creatinine content in human or animal plasma, serum, and urine samples. This product is for research use only and is not suitable for clinical diagnosis or other purposes.

C1505879	Component	100T	Storage
C1505879A	Creatinine Standard (10 mmol/L)	1 mL	2-8℃. Store in the dark.
C1505879B	Creatinine Standard Diluent	2 mL	RT
C1505879C	Cr Chromogenic Solution	10 mL	RT
C1505879D	Cr Assay Buffer	10 mL	RT

Required Materials Not Provided

1. Distilled water

2. Microplate reader, 96-well plate, Incubator or water bath

Experimental Procedure

1. Sample Preparation

• Prepare plasma and serum by conventional methods. Store at -20°C.

• If the creatinine concentration in urine is high, dilute it 1:10 to 1:100 with distilled water before assay. Excessively high dilution factors are generally not recommended as they may lead to small absorbance differences.

2. Preparation of Standard Working Solution

• Mix the Creatinine Standard (10 mmol/L) with the Creatinine Standard Diluent at a 1:49 ratio to achieve a concentration of 200 μmol/L. This is the Creatinine Standard Working Solution (200 μmol/L).

• Store at 4°C; stable for 1 week.

3. Preparation of Cr Chromogenic Working Solution

• Mix equal volumes of Cr Chromogenic Solution and Cr Assay Buffer.

• Allow the mixture to stand at room temperature for 20 minutes. This is the Cr Chromogenic Working Solution.

4. Cr Assay Setup

• Set up Standard and Test wells according to the table below.

• Add reagents in the specified order, avoiding bubbles.

• If the sample Cr concentration is too high, dilute it appropriately (10-100 fold) before assay.

Reagent (μL)	Standard Well	Test Well
Creatinine Standard (200 μmol/L)	20	—
Serum, Plasma, Diluted Urine	—	20
Cr Chromogenic Working Solution	200	200

• Note: For samples with many interfering substances, increasing the concentration of the creatinine standard (e.g., 300-400 μmol/L) can reduce error. In this case, adjust the calculation formula accordingly (multiply by 300-400 instead of 200).

• Generally, add the standard and test samples first, then add the Cr Chromogenic Working Solution, starting accurate timing and reading immediately after addition.

5. Cr Measurement

• React at 37°C.

• Mix thoroughly immediately after adding the working solution.

• Precisely at 20 seconds, measure the absorbance at 510 nm (recorded as A_standard1 and A_test1).

• Precisely at 60 seconds, measure the absorbance again (recorded as A_standard2 and A_test2).

• Note: When using a microplate reader, a clinical chemistry analyzer is recommended. Manual or semi-manual operation may be inaccurate or yield suboptimal results.

6. Calculation

• Creatinine (μmol/L) = [(A_test2 - A_test1) / (A_standard2 - A_standard1)] × 200 × N

Parameter Definitions:

• A_test1: Absorbance of Test well at 20 seconds

• A_test2: Absorbance of Test well at 60 seconds

• A_standard1: Absorbance of Standard well at 20 seconds

• A_standard2: Absorbance of Standard well at 60 seconds

• N: Sample dilution factor

Reference Interval:

Adult Males	62~115μmol/L (0.7-1.3mg/dl)
Adult Females	53~97μmol/L(0.6-1.1mg/dl)

7.  Standard Curve Reference

• Typical ΔA for 200 μmol/L Creatinine Standard: The difference in absorbance between 60s and 20s (ΔA) for a 200 μmol/L creatinine standard typically ranges from 0.02 to 0.05 when measured at 505 nm.

• A standard curve can be constructed by measuring the ΔA values for creatinine standards at 0, 50, 100, 200, 300, and 400 μmol/L.

Note: Reference ranges may vary due to differences in instruments and operational techniques. For precise creatinine quantification, use a multi-point standard curve.

Precautions

1. This assay is based on the rate method; the entire reaction is fast (approx. 2-3 min). Precise timing is critical; failure to adhere to timing may result in undetectable differences.

2. The linear range of this rate method extends up to 2000 μmol/L. Dilute samples with high concentrations before assay.

3. Ensure both samples and standards reach room temperature before assay, as temperature affects results.

4. Slight hemolysis does not affect the creatinine assay.

5. Avoid processing too many samples simultaneously to prevent inaccuracies from prolonged reagent addition times. After adding samples, add the Cr Chromogenic Working Solution to a maximum of 2-3 tubes at once. A multi-channel pipette can slightly increase this number.

6. Urine creatinine levels are generally high. If the absorbance after color development still exceeds the linear range (2000 μmol/L), dilute the urine sample 10-100 fold with distilled water and repeat the assay.

7. For your safety and health, wear lab coats and disposable gloves during operation.

8. Use reagents promptly after opening to avoid affecting subsequent experimental results.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. C1505879A: Store at 2-8℃ long term (12 months). Store in the dark. C1505879B, C1505879C, C1505879D: Store at room temperature long term (12 months).