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Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof

A technology of free thyroxine and nano-magnetic particles, which is applied in the direction of chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, etc., can solve the cumbersome preparation process of free thyroxine, poor process stability, and limitations Detection effect and other problems, to achieve the effect of good accuracy, simple operation and time-saving, and improve the detection effect

Active Publication Date: 2013-04-24
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the kit can achieve fast and accurate detection, the preparation process of free thyroxine labeled with horseradish peroxidase is very cumbersome, the process stability is not good, and the labeling rate is low, which limits its detection effect, especially for analysis. inter-precision and lead to increased cost

Method used

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  • Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof
  • Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof
  • Free thyroxine nanometer magnetic particle chemiluminescence assay kit and preparation method thereof and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1 The preparation of the first reagent

[0038] (1) Materials and instruments: anti-FT4 monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC), sodium bicarbonate and other reagents should reach the chemical Pure; G-25 gel purification column was purchased from GE.

[0039] (2) Preparation steps:

[0040] ① Use 0.1~0.2mol / L carbonate buffer solution with pH9.0~10.0 to prepare 0.5mg / mL FITC solution;

[0041] ② Add the FITC solution prepared in step ① to the antibody solution according to the ratio of FT4 antibody to FITC molecular ratio of 1:20, mix well, and let stand at room temperature for 12 hours to generate FT4 antibody-FITC conjugate;

[0042] ③Separate the reaction liquid after step ② through G-25 gel column, remove unreacted FITC, and obtain a solution containing FT4 antibody-FITC conjugate (ie, FITC-labeled FT4 antibody);

[0043] ④Dilute the solution containing the FT4 antib...

Embodiment 2

[0044] Embodiment 2 Preparation of the second reagent

[0045] (1) Materials and instruments: FT4 antigen (solid powder, purity over 95%); alkaline phosphatase preserved in phosphate buffer (ALP solution, ALP purity is about 99%, specific activity is about 1500U / mg, concentration is 10 mg / mL); the coupling agent DSS was purchased from THERMO Company, and chemical reagents such as TRIS should be chemically pure; the G-25 gel purification column was a product of GE Company.

[0046] (2) Preparation steps:

[0047] ① Take 1mg of FT4 antigen, add DMSO to dissolve the antigen to a concentration of 20-50mg / mL, add DSS0.5mg, react at room temperature for 2 hours, dilute the reaction solution 1:10 with DMSO, and store it at 2-8°C for later use;

[0048] ②Take 1mg of ALP solution, with 0.1M NaHCO pH9.5 3 Buffer Dilute the ALP solution to 1mg / mL, add the FT4-DMSO solution prepared in step ① to the diluted ALP buffer for ligation reaction, add the FT4-DMSO solution volume to 1 / 20 of th...

Embodiment 3

[0050] The preparation of embodiment 3 magnetic separation reagents

[0051] (1) Materials and instruments:

[0052] Suspension of magnetic particles: the content of magnetic particles is 5wt%, and the magnetic particles contain carboxyl (COOH) active groups. The carboxyl group content per gram (g) of magnetic particles (dry weight) is not less than 0.4 millimoles (mmol), with superparamagnetism, Diameter between 0.5-2μm

[0053] Anti-FITC antibody: It can be a polyclonal antibody or a monoclonal antibody, with a purity of more than 90% by weight and a dilution titer of more than 1:1 million;

[0054] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0055] (2) Preparation steps:

[0056] ①Take 100mg of magnetic particle suspension, magnetically separate to remove the supernatant, and resuspend in 10mL of 0.05mol / L, pH4.5~5 MES buffer;

[0057] ②Add 2~4mg of anti-FITC antibody and suspend at room temperature...

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Abstract

The invention relates to a free thyroxine nanometer magnetic particle chemiluminescence assay kit and a preparation method thereof and a detection method thereof. The free thyroxine nanometer magnetic particle chemiluminescence assay kit comprises solutions containing fluorescein-labeled free thyroid antibodies, suspension liquid of magnetic particles covered with fluorescein-resistance antibodies and solutions containing alkaline-phosphatase-labeled free thyroxine antigens, wherein the alkaline-phosphatase-labeled free thyroxine antigens are formed by the connection of alkaline phosphatase and the free thyroxine antigens through crosslinking agent suberic acid succinimide ester. The free thyroxine nanometer magnetic particle chemiluminescence assay kit and the preparation method thereof and the detection method thereof have the advantage of enabling the free thyroxine to be quantificationally detected on the condition of lower cost, higher accuracy and higher precision.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a nano-magnetic particle chemiluminescence assay kit for free thyroxine that combines immunomagnetic particle separation technology and nano-magnetic particle chemiluminescence assay technology and a preparation method thereof. The present invention also particularly relates to the preparation method of the kit. Background technique [0002] Thyroxine, 3,5,3',5'-tetraiodothyronine (T4) is a thyroid hormone with a molecular weight of 777. T4 has two forms of bound state and free state in circulation, and under normal circumstances, a dynamic balance is maintained between the two forms. 75% of circulating T4 is bound to thyroid-binding globulin (TBG), 15% to prealbumin, and 10% to albumin. Free T4 (FT4) accounts for only 0.03%, but it plays a real physiological role. Pregnancy, hepatitis, congenitally increased TBG, taking estrogen (oral contraceptives), etc. cause the TB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/78G01N21/76
Inventor 于大为程晓蕾李冬冬
Owner SUZHOU HAOOUBO BIOPHARML
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