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Dissociation method of 25-hydroxyvitamin D binding protein

A hydroxyvitamin and binding protein technology, applied in the field of vitamin D detection, can solve the problems of poor dissociation effect, toxicity, poor stability, etc., and achieve the effects of improving detection results, strong reducibility and improving stability

Inactive Publication Date: 2020-02-04
重庆黄嘉同怡科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The current dissociation methods mainly include protein denaturation method, adding denaturants to the sample solution, commonly used denaturants mainly include guanidine hydrochloride, urea or sodium dodecylsulfonate, but these denaturants are toxic and have poor stability , the dissociation effect is not good, and the overall vitamin D status cannot be accurately determined

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The operator prepares mother liquor in advance, selects dithiothreitol with a concentration of 0.01g / ml, mixes dithiothreitol with water to form a 1mol / L dithiothreitol aqueous solution, and configures multiple mother liquors, each with a capacity of 1ml, the prepared mother solution is stored for later use, the spare mother solution is stored at -20°C for short-term use, and the mother solution is stored at -80°C for long-term storage; take 200 μL serum sample, add 20 μL Mother liquor, vortex 1min to mix, then put the mixture into a centrifuge for 5min, centrifuge at 13000rpm, temperature 4°C, centrifuge for 10min under this condition; take the supernatant of the obtained solution, add 200μL ethanol , manually vortexed for 1min to mix, and the mixed solution was left to stand for 5min; the obtained solution was put into a centrifuge with a centrifugal speed of 13000rpm and a temperature of 4°C, and centrifuged for 10min under these conditions. After the centrifugation w...

Embodiment 2

[0022] The operator prepares mother liquor in advance, selects dithiothreitol with a concentration of 0.015g / ml, mixes dithiothreitol with water to form a 1mol / L dithiothreitol aqueous solution, and configures multiple mother liquors, each with a capacity of 1ml, the prepared mother solution is stored for later use, the spare mother solution is stored at -20°C for short-term use, and the mother solution is stored at -80°C for long-term storage; take 250 μL of serum samples, add 25 μL of mother solution , vortexed for 1 min to mix, and then put the mixture into a centrifuge for 5 min to centrifuge at a speed of 13000 rpm and a temperature of 4 °C for 10 min under these conditions; take the supernatant of the obtained solution, add 250 μL of ethanol, Manually vortex for 1 min to mix well, then let the mixed solution stand for 5 min; put the obtained solution into a centrifuge with a centrifugal speed of 13,000 rpm and a temperature of 4°C, and centrifuge for 10 min under these co...

Embodiment 3

[0024] The operator prepares the mother liquor in advance, selects dithiothreitol with a concentration of 0.03g / ml, mixes dithiothreitol with water to form a 1mol / L aqueous solution of dithiothreitol, configures multiple mother liquors, and the capacity of each mother liquor is 1ml, the prepared mother solution is stored for later use, the spare mother solution is stored at -20°C for short-term use, and the mother solution is stored at -80°C for long-term storage; take 300 μL serum sample, add 30 μL Mother liquor, vortex 1min to mix, then let the mixture stand for 5min, put it into a centrifuge, centrifuge at 13000rpm, temperature 4°C, centrifuge for 10min under this condition; take the supernatant of the obtained solution, add 300μL ethanol , manually vortexed for 1min to mix, and the mixed solution was left to stand for 5min; the obtained solution was put into a centrifuge with a centrifugal speed of 13000rpm and a temperature of 4°C, and centrifuged for 10min under these con...

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Abstract

The invention discloses a dissociation method of 25-hydroxyvitamin D binding protein. The dissociation method comprises the following steps: preparing a mother solution, taking a serum sample, addingthe mother solution, carrying out vortex mixing for 1min, uniformly mixing the obtained solution, allowing the obtained mixed solution to stand for 5min, and centrifuging the mixed solution in a centrifuge; and taking the obtained supernatant of the solution, adding ethanol, carrying out vortex mixing, allowing the obtained mixed solution to stand, placing the obtained solution in the centrifuge,centrifuging the solution, taking the obtained supernatant, placing the supernatant in a new centrifuge tube, blow-drying the supernatant by using a nitrogen blow-drying instrument, adding a separation mobile phase, re-dissolving the dried supernatant, uniformly mixing the re-dissolved supernatant and the separation mobile phase, and carrying out LC-MS / MS detection. The solution uses dithiothreitol with a certain concentration, so the 25-hydroxyvitamin D to be analyzed can be well separated, the detection result of the 25-hydroxyvitamin D can be improved, and the overall content of the vitaminD is further determined; and the dithiothreitol has strong reducibility, so the stability of the 25-hydroxyvitamin D in the purification and extraction process is improved.

Description

technical field [0001] The invention relates to the detection field of vitamin D, in particular to a dissociation method for 25-hydroxyvitamin D binding protein. Background technique [0002] 25-OH-VD is the main storage form of vitamin D in the human body, therefore, the overall vitamin D status can be determined by detecting 25-OH-VD. The clinical application of 25-OH-VD detection is mainly related to the diagnosis, treatment and monitoring of rickets (children), osteomalacia, postmenopausal osteoporosis and renal bone disease. Vitamin D deficiency has also been linked to many other diseases, including cancer, cardiovascular disease, autoimmune disease, diabetes and depression. [0003] Vitamin D in the body is transported to the liver through the blood through the combination with albumin and vitamin D binding protein, and with the participation of 25-hydroxylase (25-hydroxylase, CYP2R1, CYP27A1) secreted by the liver, the hydroxylation process at position 25 is complete...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 王伟
Owner 重庆黄嘉同怡科技有限公司
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