Synthetic process for alkaline phosphatase marked free thyroid hormone

A technology for thyroid hormone and synthesis process, which is applied in the field of synthesis technology of alkaline phosphatase-labeled free thyroid hormone, can solve the problems of high price of instruments and reagents, and achieve the effects of low cost and small loss of activity

Active Publication Date: 2005-10-19
CHEMCLIN DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the equipment and reagents are expensive, especially the labeling process of alkaline phosphatase (Apase) in the kit is a highly confidential proprietary technology of foreign manufacturers. Up to now, there have been no reports of this process at home and abroad.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the preparation of alkaline phosphatase polymer

[0031] (1) Take 5 mg of alkaline phosphatase (sigma product P5521) and dialyze it with normal saline for 20 hours, add normal saline to 1.0ml, add 150 μL of coupling agent glutaraldehyde dropwise, react at room temperature for 1 hour, and put it into a dialyzer with a diameter of 8 mm. In the bag, dialyze with normal saline at 4°C for 18 hours, and change the solution twice in the middle. Add the reaction solution to a 1.0×35cm SephadexG100 column pre-balanced with 20mmol / L pH 7.0 PBS, rinse with 20mmol / L pH 7.0 PBS, flow rate 0.2ml / min, collect 1 tube per 0.5mL, collect about 20 tubes in total, Use ultraviolet 280nm to detect each tube, divide the absorption peak at 280nm into 3 parts, measure the OD value on the ultraviolet spectrophotometer respectively, calculate the protein amount of each of the 3 parts, and add the same amount of AR grade glycerol to each part, 4 Store at ℃.

[0032] (2) Take 200 μg...

Embodiment 2-3

[0033] Embodiment 2-3: the preparation of alkaline phosphatase polymer

[0034] Prepare the alkaline phosphatase polymer according to the method of implementation 1, except that 100 μL and 250 μL of the coupling agent glutaraldehyde are added, the reaction time at room temperature is 2 hours, and the washing flow rate is 0.1ml / min and 0.4ml / min.

Embodiment 4

[0035] Embodiment 4: Preparation of alkaline phosphatase polymer-free thyroid hormone

[0036] (1) Take 1 mg (i.e. 2 nmol) of the alkaline phosphatase polymer obtained in Example 1 and put it into a dialysis bag, dialyze the normal saline for 6 hours, change the liquid once in the middle, add normal saline to 1000 μ L, add 10 nmol free thyroid hormone 100 μ L, Mix well, add 60 μL coupling agent glutaraldehyde dropwise to the mixture, and react at room temperature for 2.5 hours;

[0037] (2) Add the reaction solution to a 1.0×35cm Sephadex G100 column pre-balanced with 0.1mol / L pH 7.5 Tris-HCl buffer, rinse with 0.1mol / L pH 7.5 Tris-HCl buffer, flow rate 0.2ml / min , collect eluent per 0.5mL1 tube, select the peak eluent with the second absorption peak of UV 280nm;

[0038] (3) Transfer the collected eluent into a clean glass bottle, add MgCl 2 and ZnCl 2 Make its concentration 2mmol / L and 0.2mmol / L, then add NaN 3 Wagyu bovine serum albumin (BSA) was used to make the concen...

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PUM

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Abstract

The present invention discloses synthesis process of alkaline phosphatase marked free thyroid hormone. The structure of alkaline phosphatase is reformed for coupling with thyroid hormone to prepare marker antigen. The present invention has simple technological process and low cost, and solves the difficult problem of preparing alkaline phosphatase marker as the key component for free thyroid hormone detecting kit in the immunological analysis of chemical luminescence.

Description

technical field [0001] The invention relates to a synthesis process of alkaline phosphatase-labeled free thyroid hormone applied in chemiluminescence immunoassay technology. Background technique [0002] Thyroid hormones include thyroxine (T4) and triiodothyronine (Triiodothyronine, T3), most of which exist in the form of protein binding in plasma, and only less than 0.2% are in the form of free thyroxine (Free Thyronine). Thyroxine, FT4) and free triiodothyronine (FT3) exist in the form, but only the free form has physiological activity, so free triiodothyronine (FT3) and free thyroid hormone (FT4 ) is an important indicator for the diagnosis of thyroid disease. [0003] The most commonly used radioimmunoassay (RIA) is the 125 I labeled analogs to achieve, the synthesis process is complex, the validity period is short, there is some pollution to the environment, and there are many factors that affect the test results. In the past 10 years, breakthroughs have been made in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/13C07K14/575
Inventor 胡国茂李振甲马世俊
Owner CHEMCLIN DIAGNOSTICS CO LTD
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