Application

This kit can be used to measure the lactic acid (LA) content in whole blood samples. 

Detection significance

Lactic acid is the intermediate product of glucose metabolism in vivo, which is mainly produced by erythrocytes, rhabdomytes and brain tissues. The concentration of lactate in blood depends on the synthesis speed and metabolic rate of liver and kidney. The bidirectional transformation of lactic acid and pyruvate is regulated by lactate dehydrogenase (LDH).

Detection principle

Using NAD+ as H+ receptor, LDH catalyzes the conversion of both lactate and NAD+ into pyruvic acid and NADH respectively. 1-Methoxy-5-methyl phenazine methyl sulfate (PMS) transfers hydrogen from NADH to NBT which deoxidize into purple chromogenic substrate. Lactic acid content can be calculated by measuring the OD value at 530 nm.

Experimental instrument

Tube, Micropipette, Vortex mixer, Centrifuge, Spectrometer (530 nm), Water bath

Operation steps

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment.

1. The pretreatment of sample:

Add Reagent 1 to whole blood sample according to the ratio of whole blood (mL): Reagent 1 (mL)=1: 6 (for example, add 0.3 mL Reagent 1 to 0.05 mL whole blood sample). Then centrifuge at 3100 g for 10 min. Take the supernatant for detection.

2. Assay steps:

(1) Blank tube: add 20 μL of double distilled water into a 5 mL EP tube.

Standard tube: add 20 μL of 3 mmol/L Standard into a 5 mL EP tube.

Sample tube: add 20 μL of sample into a 5 mL EP tube. 

(2) Add 1000 μL of Enzyme working solution and 200 μL of Reagent 4 and oscillate fully.

(3) Incubate the tubes at 37℃ water bath for 10 min.

(4) Add 2000 μL of Reagent 6 working solution and mix fully.

(5) Set the Spectrometer to zero with double distilled water and measure the OD value of each tube at 530 nm with 1 cm diameter cuvette.

Note: It can be refer to the following operating table.

Technical parameters

1. The sensitivity of the kit is 0.14 mmol/L.

2. The intra-assay CV is 1.6% and the inter-assay CV is 2.0%.

3. The recovery of the kit is 101%.

4. The linear range of the kit is 0.14-7.0 mmol/L.

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The valid of kit is 6 months.

4. Do not use components from different batches of kit.