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Pretreatment method and detection method for measuring concentration of transthyretin (TTR) tetramer in blood

A technology for determination of thyroxine protein and concentration, applied in the field of analysis and detection, can solve the problems of inability to obtain TTR, inability to distinguish the type of TTR tetramer, inability to measure TTR tetramer alone, etc., and achieves short detection time, good specificity, Simple to use effects

Active Publication Date: 2022-04-12
大连博源医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method measures the total TTR concentration in the blood, including TTR monomers, dimers, and tetramers, which is the sum of these three concentrations. It cannot specifically measure the concentration of TTR tetramers alone, nor can it obtain Amount of misfolded TTR, more indistinguishable from TTR tetramer type

Method used

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  • Pretreatment method and detection method for measuring concentration of transthyretin (TTR) tetramer in blood
  • Pretreatment method and detection method for measuring concentration of transthyretin (TTR) tetramer in blood
  • Pretreatment method and detection method for measuring concentration of transthyretin (TTR) tetramer in blood

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Embodiment 1

[0048] Example 1 Determination of the concentration of transthyretin (TTR) tetramer in 96 cases of plasma samples

[0049] Plasma samples were collected from apparently healthy people aged 20-60 years old collected by Dalian Runsheng Kangtai Medical Laboratory Co., Ltd., including 49 males and 47 females, all of whom had signed informed consent.

[0050] 1 Configuration of calibration solution

[0051] (1) Preparation of calibration working solution: Accurately prepare recombinant wild-type transthyretin (TTR) tetramer stock solution (80 μmol / L), dilute it with PBS (pH 7.4) to obtain an intermediate stock solution, and use interval dilution method Dilute the intermediate stock solution to obtain a series of transthyretin (TTR) tetramer calibration working solutions with a concentration of 1 μmol / L, 2 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L, for future use.

[0052] (2) Calibration curve solution: Take 50 μL of calf serum (blank matrix), add 50 μL of recombinan...

Embodiment 2

[0081] Example 2 Qualitative identification and concentration determination of transthyretin tetramer in plasma

[0082] A total of four experimental samples were obtained from Dalian Runsheng Kangtai Medical Laboratory Co., Ltd. Among them, there were 3 genetic samples of the D39Y variant family (father and two daughters), of which sample 1 was the father, who had been diagnosed by a local tertiary hospital. Transthyretin amyloid cardiomyopathy (ATTR-CM); sample 2 is the eldest daughter, who was identified as a gene carrier by gene sequencing in a local tertiary hospital; sample 3 is the second daughter, who was identified as a gene carrier by gene sequencing in a local tertiary hospital normal sample. Sample 4 is a patient with T96R mutation, who was identified as a gene carrier by gene sequencing in a local tertiary hospital, and the above four candidates have signed informed consent.

[0083] 1. Configuration of calibration solution

[0084] Except that the recombinant...

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Abstract

The invention discloses a pretreatment and detection method for determining concentration of TTR (transthyretin) tetramer in blood, which comprises the following steps: adding a small-molecule fluorescent probe after the blood passes through a membrane, and incubating to enable the small-molecule fluorescent probe to be specifically combined with TTR to generate fluorescence; and adding a diluent into the incubated sample, diluting, centrifuging, taking supernate, separating by adopting high performance liquid chromatography, and carrying out quantitative analysis by utilizing a fluorescence detector. The method is simple and convenient to operate, the concentration of the TTR tetramer in the blood can be quantitatively measured, the TTR misfolding concentration can be obtained by subtracting the concentration of the TTR tetramer from the total concentration of the TTR, whether the TTR tetramer is of a wild type or a variant type can be qualitatively identified through retention time, and the method has good selectivity, precision and accuracy.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a pretreatment method and a detection method for detecting the concentration of transthyretin (TTR) tetramers in blood samples. Background technique [0002] Prealbumin, also known as transthyretin (TTR for short), is a tetrameric protein composed of 127 amino acids and a molecular weight of 55KDa. It is mainly produced by the choroid plexus of the liver and brain, and a small amount is synthesized by retinal epithelial cells, and then secreted into the blood and cerebrospinal fluid. It has the functions of transporting thyroid hormone and binding RBP protein. [0003] Usually, TTR is composed of four identical monomers. In ATTR amyloidosis (ATTRAmyloidosis), the stability of the tetrameric protein decreases, and the protein breaks down into dimers and monomers, showing a partially unfolded conformation, and automatically Assembled into toxic non-fibri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/14G01N30/88G01N30/86
Inventor 刘宇曹云峰崔丽关丽萍刘明莉崔雪婷高鹏
Owner 大连博源医学科技有限公司