檸檬酸檢測(cè)試劑盒

使用說(shuō)明書

僅供體外研究使用，不用于臨床診斷!

第1版（2016年04月修訂）

[ INTENDED USE ]

The kit is a colorimetric method for the in vitro quantitative measurement of Citric Acid in samples.

Citric acid (CA), an organic acid commonly found in living organisms, is an important food flavoring substance. In addition, CA is the product of the first reaction of the tricarboxylic acid cycle.

[ REAGENTS AND MATERIALS PROVIDED ]

[ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1. A visible spectrophotometer capable of measuring absorbance at 545nm

2. Thermostatic water bath capable of controlling temperature at 30℃

3. High-speed freezing centrifuge

4. Micropipets and tips

5. 1 mL glass cuvette

6. Distilled water (preferably double distilled water and double distilled water)

[ STORAGE OF THE KITS ]

Reagent 1: liquid. Can be stored at 4℃ for 6 months.

Reagent 2: liquid. Can be stored at 4℃ for 6 months.

Reagent 3: liquid. Can be stored at -20℃ for 6 months.

       Reagent 4: powder. Can be stored at room temperature for 6 months.

Reagent 5: liquid. Can be stored at -20℃ for 6 months. Away from light.

Standard: 250μmol/L Citric acid standard solution, liquid. Can be stored at 4℃ for 6 months.

[ REAGENT PREPARATION ]

Reagent 4 Preparation: Before use, add 5ml reagent 1 to dissolve the powder completely.

[ SAMPLE PREPARATION ]

1.  Citric acid extraction from liquid sample

Take 0.1 ml of liquid and add 0.9ml of reagent 1, mix well. 11000 g, centrifuge the sample at 10℃, 11000g for 10 min. Extract the supernatant on ice for further measurement.

2.  Citric acid extraction from tissue sample

Weight the sample precisely and add the reagent 1 with ratio of 1g sample with 4-9ml of reagent 1(w:v = 1:5-1:10, e.g. 1mL of reagent 1 is added in 0.1g tissue sample). Homogenize the mixture in the ice water bath and the centrifuge the homogenate at 4℃, 11000 g for 10min. Extract the supernatant on ice for further measurement. 

3.  Citric acid extraction from mitochondria sample

Weight the sample precisely and add the reagent 1 with ratio of 1g sample with 4-9ml of reagent 1(w:v = 1:5-1:10, e.g. 1mL of reagent 1 is added in 0.1g tissue sample). Homogenize the mixture in the ice water bath and the centrifuge the homogenate at 4℃, 600 g for 5min. Collect the supernates in another EP tube and centrifuge it at 4℃, 11000 g for 10min. Remove the supernatant and then add 200ul of reagent 2 and 2ul of reagent 3. after    on ice for further measurement. Mix sufficiently and centrifuge it at 4℃, 11000 g for 10min. Extract the supernatant on ice for further measurement. 

4.  Citric acid extraction from bacteria and fungi sample.

Add the reagent 1 with ratio of 500-1000×10⁴ cells with 1ml of reagent 1(e.g. 1mL of reagent 1 is added in 500×10⁴ cells). The cells could be subjected to ultrasonication till the solution is clarified(e.g. Ultrasonic power: 300W, ultrasonic:3 seconds, Stop:7 seconds, total time: 3 minutes). Centrifuge at 4℃, 11000g for 10 minutes to remove cellular debris. Extract the supernatant on ice for further measurement. 

[ ASSAY PROCEDURE ]

Operation table:

Note: Before use, the reagent should be preheated in a 30℃ water bath for 30 min.

[ TEST PRINCIPLE ]

Under acidic conditions, citric acid reduced Cr6+ to form Cr3+, and Cr3+ had a characteristic absorption peak at 545nm. By measuring the increase of 545nm absorbance, the citric acid content in the sample can be calculated.

[ CALCULATION OF RESULTS ]

[ IMPORTANT NOTE ]

1.  Samples pre-treatment should be carried out on ice.

2.  Prepare reagent 4 within 15 minutes before assay. The reconstituted reagent 4 can be used only once.

3.  Reagent 5 contains carcinogens. During the experiment, wear gloves to prevent skin exposure.

4.  The citric acid extract buffer can not be used for the determination of protein concentration. If needed, it is recommended to take an additional tissue and use IS003 BCA Protein Quantification Kit to determine the protein concentration.