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Quick magnetic method for separating and purifying thyroxin marked-by biotin

A technology of biotin labeling and thyroxine, applied in the field of biochemistry, can solve problems such as unsuitable for large-scale separation operations, limited reaction volume, etc.

Inactive Publication Date: 2004-10-27
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the separation and purification method of immobilizing bovine serum albumin with an enzyme-labeled orifice plate has a limited reaction volume and is not suitable for large-scale separation operations.

Method used

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  • Quick magnetic method for separating and purifying thyroxin marked-by biotin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] (1) Biotin labeling reaction of thyroxine

[0029] Dissolve biotin N-hydroxysuccinimide ester in dimethyl sulfoxide to make a 0.5mg / ml solution; dissolve thyroxine in DMF to make a 2mg / ml stock solution, use 0.1M, pH8.5 for use NaHCO 3 The buffer was diluted to 0.2 mg / ml. Take 100 μl of 0.5 mg / ml biotin N-hydroxysuccinimide ester solution and slowly add to 100 μl of 0.2 mg / ml thyroxine solution, and shake for 2 hours at room temperature.

[0030] The molar ratio of biotin N-hydroxysuccinimide ester to thyroxine is about 5:1. After 2 hours of full reaction, thyroxine was basically biotinylated. The reaction mixture contains essentially only biotin-labeled thyroxine and excess biotin N-hydroxysuccinimide ester. The total concentration of thyroxine is 0.1mg / ml.

[0031] (2) Magnetic separation to remove excess biotin N-hydroxysuccinimide ester, and enzyme immunoassay to detect the reaction degree of biotin N-hydroxysuccinimide ester with bovine serum albumin on the magn...

Embodiment 2

[0044] The reaction molar ratio of biotin N-hydroxysuccinimide ester and thyroxine was changed to 3:1, the separation and detection method was the same as in Example 1, and the one-step method was used for separation and detection. The detection results of the four batches of magnetic particles taken out every 15 minutes are: 0.285, 0.455, 0.579, 0.581. It shows that the amount of bound biotin N-hydroxysuccinimide ester on the magnetic particles does not change after 45 minutes. After magnetic separation, take the supernatant and react with the magnetic particles containing bovine serum albumin. The detection value is 0.084 and the control value (0.085) close. It shows that there is no excess biotin N-hydroxysuccinimide ester in the solution.

[0045] The separated supernatant was taken to detect the biotinylation rate of thyroxine, the detection method was the same as in Example 1, and the detection value was 0.212. According to the molar concentration relationship, the cor...

Embodiment 3

[0047] The reaction molar ratio of biotin N-hydroxysuccinimide ester and thyroxine was changed to 8:1, the separation and detection method was the same as in Example 1, and a multi-step method was used for separation and detection. The detection results of the magnetic particles after the 4 batch reactions were: 0.822, 0.542, 0.247, 0.099. Explain that after the 4th reaction, the detection value on the magnetic particle is close to the control value (0.086), indicating that the excess biotin N-hydroxysuccinimide ester in the solution has completely reacted with the bovine serum albumin on the magnetic particle and passed Magnetic separation removes, that is, there is no excess biotin N-hydroxysuccinimide ester in the solution.

[0048] The separated supernatant was taken to detect the biotinylation rate of thyroxine, the detection method was the same as in Example 1, and the detection value was 0.210. According to the molar concentration relationship, the corresponding biotin...

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Abstract

After reaction between bovine serum albumin (bsa) on magnetic particles and biotin label of thyroxin, superfluous biotin reagent in mixed liquor and n-hydroxyl succimide ester generates compound of magnetic particle-bsa-biotin. Under action of external magnetic field, superfluous biotin reagent is removed. Degree of reaction between n-hydroxyl succimide ester and magnetic particles is tested by enzyme immunoassay method. It is determined that final obtained solution is high-purified thyroxin labeled by biotin. Advantages of the invention are simple, quick and high efficiency. The invention is also applicable for separating and purifying biomoleculars of protein, polypeptide, oligonucleotide etc. labeled by biotin.

Description

technical field [0001] The invention relates to a fast magnetic separation and purification method, in particular to a fast magnetic separation and purification method for biotin-labeled thyroxine. Belongs to the field of biochemistry. Background technique [0002] The biotin-avidin system is a new type of biological reaction amplification system that has developed rapidly in recent years, and has been widely used in the fields of immunology, molecular biology and clinical medicine. When using the biotin-avidin system for biological analysis, the biomolecule must be biotin-labeled, and the excess biotin reagent after the biotin labeling reaction must be separated and removed, because the excess biotin reagent will bind to the biotin-labeled substance Competes for binding avidin, making subsequent assays inaccurate. For example, when the biotin-avidin system is introduced into the determination of human serum thyroxine and the thyroxine-biotin-avidin system-ELISA method is ...

Claims

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Application Information

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IPC IPC(8): C07K1/14G01N33/531
Inventor 郑伟明高峰张玲古宏晨
Owner SHANGHAI JIAO TONG UNIV
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