Glucose-6-phosphate dehydrogenase fluorescence spot test
Glucose-6-phosphate dehydrogenase fluorescence spot test
In the presence of glucose-6-phosphate (GbB) and NADP, glucose-6-phosphate dehydrogenase (G6PD) is able to use NADP to reduce to NADPH, which does not fluoresce under UV irradiation. This experiment was obtained from Mudanjiang Medical College Undergraduate 5-year laboratory guide for testing majors
Operation method
Glucose-6-phosphate dehydrogenase fluorescence spot test
Principle
In the presence of glucose-6-phosphate (GbB) and NADP, glucose-6-phosphate dehydrogenase (G6PD) is able to use NADP to reduce to NADPH, which does not fluoresce under UV irradiation.
Materials and Instruments
G6P NADP HCl Move I. Experimental reagents: adults and formulations of mixed reagents: Caveat 1. The assay is direct; the amount of NADPH is more specific. 2. specimens from normal and defective G6PD subjects should be used as controls for each assay or batch. For more product details, please visit Aladdin Scientific website.
Oxidized glutathione Saponin Distilled water
0.1mol/L G6P 1ml
7.5mmol/L NADP 1ml 0.7mol/L tris-HCl (PH7.8) 3ml
8mmol/L oxidized glutathione 1ml
10g/L saponin 2ml
Distilled water 2ml
If the mixed reagent is stored at -20℃ after dispensing, it can be stabilized for several months
Second, the experimental operation:
1. Specimen collection: EDTA-Na2H or heparin anticoagulated whole blood, which can be stable for 1 week if stored at 4℃, or use heparinized capillary to collect peripheral blood from finger or foot staggers.
2. Take 3 test tubes of 12MM×75MM, patients, patients, patients, patients, patients, patients, patients, patients, patients, patients, patients, patients, patients, patients, patients, patients. Normal (control) and positive (control), the blood of each tube to add 200 microliters of reagent.
3. Add 20 microliters of patient's normal and positive anticoagulated whole blood to each tube, mix and place at 25℃ room temperature.
4. Pipette 1 drop of reaction solution from each tube at 0/min (immediately after mixing) 5 min and 10 min, respectively, and add it to Xinhua No. 1 liquid paper to dry thoroughly.
5. In a dark room, irradiate the spots on the filter paper with ultraviolet light at a wavelength of 200-340nm to check for fluorescence.
6. 0min spots of normal people are non-fluorescent, 5min and 10min spots appear fluorescent, 10min spots fluorescence is the strongest, G6PD-deficient patients in 0min, 5min and 10min are non-fluorescent.
Experimental results: normal people have very strong fluorescence, G6PD-deficient patients have weak or no fluorescence, heterozygous or some G6PD variants may have mild to moderate fluorescence.