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Kit used for detecting enzyme activity of biotin enzyme

A technology of biotinidase and kit, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of short enzyme activity stability time, large sample consumption, and inability to apply newborn screening, etc., so as to be easy to popularize and use, meet The effect of timely diagnosis and treatment

Inactive Publication Date: 2016-04-20
北京中科非凡生物技术有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the method established by Ebrahim et al. can only be used for the detection of serum samples, the sample volume is large, the enzyme activity stability time is short, and the sample must be dialyzed before detection, which is complicated to operate.
The above deficiencies prevent this method from being applied to newborn screening, but as mentioned above, biotinidase deficiency must be diagnosed and treated in time, otherwise irreversible damage will occur

Method used

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  • Kit used for detecting enzyme activity of biotin enzyme
  • Kit used for detecting enzyme activity of biotin enzyme
  • Kit used for detecting enzyme activity of biotin enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, detection kit and application of biotinidase enzyme activity

[0064] (1) A kit for detecting biotinidase enzyme activity, comprising the following components:

[0065] Reagent 1:

[0066]

[0067] (2) Detection of biotinidase enzyme activity

[0068] 1) Sample preparation

[0069] The sample is the plasma of a healthy adult, and the dosage is 10 μL.

[0070] 2) Detection of samples

[0071] Add plasma sample to reagent 1, the volume ratio of plasma sample to reagent 1 is 1:19, add the sample and reagent and mix to make it react, react at 37°C for 24 hours, add reagent 2 to terminate the reaction after the reaction, reagent 2 and reagent The volume ratio of reagent 1 is 1:20. After termination, centrifuge the sample tube at 10000g for 10min, take the supernatant, add reagent 3, wherein the ratio of supernatant to reagent 3 is 2:1, and add reagent 4 after neutralization (pH is 9), wherein the supernatant and reagent The ratio of 4 was 1:1, and the ...

Embodiment 2

[0081] Embodiment 2, the detection of biotinidase enzymatic activity

[0082] Volunteers with biotinidase deficiency confirmed by clinical diagnosis and genetic analysis were used as the analysis objects, and healthy volunteers were used as the control.

[0083] The sample is a dried blood filter paper, the same as in Example 1.

[0084] The detection method was the same as that in Example 1, and each sample was subjected to 3 independent enzyme activity determination experiments, and the detection results are shown in Table 1.

[0085] Table 1 The average value of enzyme activity of healthy adults and patients with biotinidase deficiency

[0086]

[0087] As can be seen from the data in Table 1, the enzyme activity of healthy adult samples is: 102.65 ± 9.17 (n=10); the enzyme activity of patients with biotinidase deficiency is only 0.89% of the healthy adult control group, this result is consistent with the conclusion of gene analysis match. It has been verified that th...

Embodiment 3

[0088] Embodiment 3, the detection of biotinidase enzymatic activity

[0089] The sample is a dried blood filter paper, the same as in Example 1.

[0090] The detection method is the same as in Example 1. A total of 6 groups of reactions are set up, each group contains 2 samples and 2 blank controls, and a group of reactions is terminated at 4h, 7h, 16h, 20h, 24h and 30h respectively, and then the derivation reaction is carried out , and detect the fluorescence value to investigate the stability of biotinidase in the dried blood film during the reaction (such as figure 2 As shown, the linear equation is: y=73.97x+66.64, R 2 = 0.998). from figure 2 It can be seen that within 30 hours, the biotinidase enzyme activity in the dried blood film is stable, and the detection result is reliable.

[0091] As can be seen from the foregoing examples, conventional fluorescent detection equipment can be used to detect biotinidase enzyme activity using the kit of the present invention,...

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Abstract

The invention discloses a kit used for detecting enzyme activity of biotin enzyme. The kit comprises a reagent 1, a reagent 2, a reagent 3 and a reagent 4 which are independently packaged. The reagent 1 is a buffer system of a buffer 1, an enzyme activity stabilizing agent and biocytin, a pH value of the reagent 1 is 3-11; the reagent 2 is a stopping solution; the reagent 3 is a neutralization solution; the reagent 4 is a system composed of the buffer 2 and a derivative substrate, and the derivative substrate is any one of 1,2-diacetybenzene, ninhydrin, fluorescamine and o-phthalaldehyde. When the kit is used for detection, dry blood slices can be taken as a detection sample, so that the method can be used for neonatal screening, can satisfy timely diagnosis and treatment requirements of biotin enzyme deficiency, and is easily popularized and used.

Description

technical field [0001] The invention relates to a kit for detecting enzyme activity, in particular to a kit for detecting enzyme activity of biotinidase, which belongs to the field of biomedical detection. Background technique [0002] Biotin (Biotin) also known as vitamin B 7 , vitamin H, and coenzyme R are water-soluble sulfur-containing vitamins. Biotin is the coenzyme of four carboxylases in the body, including pyruvate carboxylase, the key enzyme in sugar metabolism, acetyl-CoA carboxylase, which catalyzes the first step of fatty acid synthesis, and propionyl, which plays an important role in protein metabolism. CoA carboxylase and β-methylcrotonyl-CoA carboxylase. The lack of biotin will lead to the loss of the above-mentioned enzyme activity, causing abnormal metabolism of the three major nutrients in the body, sugar, fat and protein, and mitochondrial energy synthesis disorders, causing metabolic acidosis, organic aciduria and a series of nerve and skin system dama...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCG01N33/573
Inventor 郝淑静刘晓敏刘卫德胡洁
Owner 北京中科非凡生物技术有限公司
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