Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof

A nano-magnetic particle, thyroxine technology, applied in chemiluminescence/bioluminescence, biological testing, analysis by chemical reaction of materials, etc., can solve the problem of high preparation cost and use cost, limit detection method precision, and preparation process Cumbersome and other problems, to achieve the effect of low production cost, good accuracy and high sensitivity

Inactive Publication Date: 2013-04-17
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cost of preparation and use of this kit is high because, on the one hand, when preparing T4-IgG-coated magnetic particles, the magnetic particles are directly placed in T4-IgG carbonate buffer , the preparation time is long, and vacuum drying is required in the end, the preparation process is cumbersome, and the requi

Method used

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  • Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof
  • Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof
  • Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof

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Embodiment 1

[0042] Embodiment 1 The preparation of the first reagent

[0043] (1) Materials and instruments: thyroxine (T4) monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FI TC), sodium carbonate and other reagents It should be chemically pure; G-25 gel purification column was purchased from GE.

[0044] (2) Preparation steps:

[0045] ① Prepare 0.5 mg / mL FITC solution with 0.1-0.2 mol / L carbonate buffer solution with pH 9.0-10.0;

[0046] ② Add the FITC solution prepared in step ① to the antibody solution according to the ratio of thyroxine monoclonal antibody to FITC molecular ratio of 1:20, mix well, let stand at room temperature for 12 hours, and react to generate T4 antibody-FITC conjugate;

[0047] ③Separate the reaction liquid after step ② through G-25 gel column, remove unreacted FITC, and obtain a solution containing T4 antibody-FITC conjugate (ie, FITC-labeled thyroxine antibody);

[0048] ④Dilute the s...

Embodiment 2

[0049] Embodiment 2 Preparation of the second reagent

[0050] (1) Materials and instruments: T4 antigen (solid powder, purity more than 95wt%); 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 cross-linking agent DSS was purchased from THERMO Company, and chemical reagents such as TRI S should be chemically pure; the G-25 gel purification column was a product of GE Company.

[0051] (2) Preparation steps:

[0052] ① Take 1 mg of T4 antigen, add DMSO to dissolve the antigen to a concentration of 20-50 mg / mL, add DSS 0.5 mg, 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;

[0053] ②Take 1mg of ALP solution, with 0.1M NaHCO pH 9.5 3 Buffer Dilute the ALP solution to 1mg / ml, add the T4-DMSO solution prepared in step ① to the diluted ALP buffer for ligation reaction, add the T4-DMSO solution to 1 / ...

Embodiment 3

[0055] The preparation of embodiment 3 magnetic separation reagents

[0056] (1) Materials and instruments:

[0057] 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, The diameter is between 0.5-2μm.

[0058] 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;

[0059] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0060] (2) Preparation steps:

[0061] ①Take 100mg of magnetic particle suspension, magnetically separate to remove the supernatant, and resuspend in 10mL of 0.05mol / L, pH 4.5~5 MES buffer;

[0062] ②Add 2~4mg of anti-FI TC antibody and suspend at room t...

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Abstract

The invention relates to a thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as a preparation method and a detection method thereof. The kit comprises solution containing fluorescein marked thyroxine antibody, suspension of magnetic particles coated with fluorescein antibody, and solution containing alkaline phosphatase marked thyroxine antigen. The kit, the preparation method and the detection method can implement quantitative detection for the thyroxine with lower cost and higher accuracy and precision.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a thyroxine (T4) nanometer magnetic particle chemiluminescence assay kit combining immunomagnetic particle separation technology and chemiluminescence immunoassay technology, and a preparation method and detection method thereof. 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. The synthesis of thyroid hormones is regulated by many factors, the most important being the hypothalamus, pituitary, and thyroid axis. Thyrotropin-releasing hormone (TRH) secreted by the hypothalamus stimulates the pituitary gland to secrete thyrotropin (TSH), which ...

Claims

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Application Information

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IPC IPC(8): G01N33/78G01N21/76
Inventor 于大为程晓蕾
Owner SUZHOU HAOOUBO BIOPHARML
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