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Kit for detecting thyroid peroxidase antibody and subtype thereof

A peroxidase and thyroid technology, applied in the field of biomedicine, can solve the problems of poor sensitivity, repeatability and specificity, narrow linear range, short detection time, etc., and achieve the effect of strong specificity and high sensitivity

Active Publication Date: 2022-03-25
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA method is simple to operate, has a short detection time, and has no radiation pollution, but the medium used in enzyme immunoassay is toxic or carcinogenic, the sensitivity, repeatability, and specificity are poor, the linear range is narrow, and it is difficult to realize full automation and other methodological constraints; at the same time, currently There are some conflicting reports about the subclass distribution of thyroid autoantibodies determined by this method

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  • Kit for detecting thyroid peroxidase antibody and subtype thereof
  • Kit for detecting thyroid peroxidase antibody and subtype thereof
  • Kit for detecting thyroid peroxidase antibody and subtype thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] 1. Detection of thyroid peroxidase antibody and its subtype

[0026] 1. Determination of serum TPOAb total IgG:

[0027] (1) Antigen dilution: Take a 15mL centrifuge tube and dilute with 6mL of pH 7.4 antigen buffer 125 I-TPO to 20000CPM / 60μL;

[0028] (2) Incubate the labeled antigen with the serum overnight: Take a 96-well plate, add 5 μL of the serum to be tested in each well, and then add 60 μL of the diluted labeled antigen to each well. The CPM value of each well should be ≥20000. Each sample and The quality control serum is double-well, the labeled antigen and the serum are shaken and mixed at room temperature for 1 hour, and then overnight at 4°C;

[0029] (3) Incubate 96-well PVDF plate: Take a 96-well PVDF micropore filter plate, add 150 μL of antigen buffer to each well, and overnight at 4°C;

[0030] (4) Precipitate antigen-antibody complexes: Discard the 96-well PVDF plate incubation solution, add 25 μL of protein A / G-agarose to each well, and transfer 5...

Embodiment 2

[0059] Comparison of agreement and correlation with electrochemiluminescence kits

[0060] 101 human serum samples were detected simultaneously with MODMLAR ANALYTICS E170 automatic electrochemiluminescence immunoassay system and supporting kits (Roche Diagnostics, Germany). The TPOAb test results of 101 outpatients were classified into negative and positive results (Table 3), the RLA positive coincidence rate was 100%, the negative coincidence rate was 83.3%, the total coincidence rate was 96%, and the Kappa value was 0.884, suggesting that the radioligand detection method ( RLA) and ECLIA methods had a high degree of consistency, and the difference was not significant (P>0.05). Spearman correlation analysis was carried out on 75 TPOAb measurements within the detection limit range (5-600U / mL), suggesting that there was a significant positive correlation between the TGAb measured by the RLA method and the ECLIA method, and the linear correlation was very strong (r=0.775 , P<0...

Embodiment 3

[0064] Determination of each IgG subtype of TPOAb

[0065] Detection of IgG subtypes of TPOAb in serum of patients with Hashimoto's thyroiditis. According to the thyroid function status at the first diagnosis, they were divided into 3 groups: hypothyroid group, subclinical hypothyroid group (subclinical hypothyroid group), normal thyroid function group (normal thyroid function group).

[0066] figure 2 It is the distribution map of TPOAb IgG subtype in the serum of patients with Hashimoto's thyroiditis (84 cases). figure 2 The results showed that in patients with Hashimoto's thyroiditis, IgG1 and IgG4 subtypes of TPOAb were mainly distributed (IgG1%: IgG2%: IgG3%: IgG4% = 51.66: 11.93: 8.94: 23.48), and IgG1 and IgG4 The percentage of total IgG was statistically different from that of other subtypes.

[0067] Table 3 The comparison of the percentage of each IgG subtype of TPOAb in the total Ig in the hypothyroid group, hypothyroid group, and normal thyroid function group ...

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Abstract

The invention discloses a kit for detecting thyroid peroxidase antibodies and subtypes thereof, and belongs to the technical field of biological medicines. The kit comprises < 125 > I labeled human thyroid peroxidase, a streptavidin agarose gel reagent, a biotinylated anti-human IgG subtype monoclonal antibody, protein A agarose and protein G agarose. On the basis of an existing radioactive ligand detection method, core steps are replaced by combining upstream and downstream principles, the streptavidin agarose gel reagent and the biotinylated anti-human IgG subtype monoclonal antibody are introduced for the first time, a core technical platform is created again for subtype typing of the antibody, and the method has the advantages of high specificity, high sensitivity and high sensitivity. And then the biotinylated anti-human IgG subtype monoclonal antibody is combined with the subtype of the thyroid peroxidase antibody, so that the detection of the thyroid peroxidase antibody subtype is realized, and the upgrading of a technical platform and the breakthrough of a thyroid peroxidase antibody subtype detection technology are successfully realized.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a kit for detecting thyroid peroxidase antibody and its subtype. Background technique [0002] Thyroid peroxidase (TPO) is a membrane protein with a molecular weight of about 103kD that binds to heme. It is located on the top cell membrane of the thyroid follicular epithelium and is a key enzyme that catalyzes the synthesis of thyroid hormones. Thyroid peroxidase antibody (TPOAb) is one of the important autoantigen targets of autoimmune thyroid disease, which can lead to the destruction of thyroid cells through antibody and complement-dependent cell-mediated cytotoxicity, and is the hallmark antibody of Hashimoto’s thyroiditis , but different patients with the same serum TPOAb concentration have different degrees and progress of thyroid damage, and it is difficult to evaluate the severity of the disease only by detecting TPOAb titer. TPOAb is mainly immunoglobuli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/60G01N33/577G01N33/573
CPCG01N33/6854G01N33/60G01N33/577G01N33/573G01N2333/908
Inventor 郑旭琴赵成程崔岱李璐阳顾愹付煜陈恒杨涛
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
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