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

A technology of triiodothyroid and nano-magnetic particles, which is applied in the fields of chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, which can solve the problems of high preparation cost and use cost, limited detection method precision, difficult Control stability and other issues, to achieve the effect of low production cost, good accuracy, and small difference between batches of analysis

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

However, the preparation cost and use cost of the kit are high, because, on the one hand, when preparing the magnetic particle suspension coated with diiodothyronine-gelatin, not only the process is complicated, but also the two The coating rate of iodothyronine-gelatin on magnetic particles is low, resulting in higher cost; on the other hand, it adopts horseradish peroxidase-labeled free triiodothyronine antibody, which The preparation of antibodies is also very cumbersome, and the labeling rate is low, which limits its detection effect and increases costs
In addition, the uncontrollable and poor stability factors in the preparation process of the kit not only lead to the problem of increased cost as mentioned above, but also make the difference between batches of detection large, which limits the precision of the detection method

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of the first reagent

[0042] (1) Materials and instruments: anti-FT3 monoclonal antibody stored in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC), sodium bicarbonate and other reagents should reach chemical Pure; G-25 gel purification column was purchased from GE.

[0043] (2) Preparation steps:

[0044] ① Prepare 0.5mg / mL FITC solution with 0.1~0.2mol / L carbonate buffer with pH 9.0~10.0;

[0045] ②Add the FITC solution prepared in step ① to the antibody solution according to the ratio of FT3 antibody to FITC molecule ratio of 1:20, mix well, stand at room temperature for 12 hours, and react to generate FT3 antibody-FITC linker;

[0046] ③The reaction solution after step ② is separated by a G-25 gel column to remove unreacted FITC to obtain a solution containing FT3 antibody-FITC conjugate (ie FITC-labeled FT3 antibody);

[0047] ④Dilute the solution containing the FT3 antibody-FITC conjugate obtained in step ③...

Embodiment 2

[0048] Example 2 Preparation of the second reagent

[0049] (1) Materials and equipment: FT3 antigen (solid powder, purity over 95%); alkaline phosphatase stored in phosphate buffer (ALP solution, ALP purity is about 99%, specific activity is about 1500 U / mg, and the concentration is 10mg / mL); coupling agent DSS was purchased from THERMO, chemical reagents such as TRIS should be chemically pure; G-25 gel purification column is a product of GE.

[0050] (2) Preparation steps:

[0051] ①Take 1 mg of FT3 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 at 2-8°C for later use;

[0052] ②Take 1mg of ALP solution and use 0.1M pH9.5 NaHCO 3 Buffer diluted ALP solution to 1mg / mL, add FT3-DMSO solution prepared in step ① to the diluted ALP buffer for ligation reaction, add FT3-DMSO solution volume to 1 / 20 of the volume of ALP buffer, and let stand at room te...

Embodiment 3

[0054] Example 3 Preparation of magnetic separation reagent

[0055] (1) Materials and instruments:

[0056] Magnetic particle suspension: magnetic particle content 5wt%, magnetic particle contains carboxyl group (COOH) active group, carboxyl group content per gram (g) magnetic particle (dry weight) is not less than 0.4 millimoles (mmol), with super paramagnetism, The diameter is between 0.5-2μm;

[0057] Anti-FITC antibody: It can be a polyclonal antibody or a monoclonal antibody, with a purity of more than 90wt%, and a dilution titer of more than 1:1 million;

[0058] 2-Morpholine ethanesulfonic acid (MES), carbodiimide (EDC), TRIS and other reagents should be chemically pure.

[0059] (2) Preparation steps:

[0060] ① Take a suspension of 100 mg of magnetic particles, magnetically separate the supernatant, and resuspend in 10 mL of 0.05 mol / L, pH 4.5-5 MES buffer;

[0061] ②Add 2~4mg of anti-FITC antibody and suspend at room temperature for 30~60min;

[0062] ③Add 0.5~1mL of a freshly ...

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Abstract

The invention relates to a free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and a preparation method thereof and a detection method thereof. The free triiodothyronine nanometer magnetic particle chemiluminescence assay kit comprises first reagents, second reagents and magnetic separation reagents, wherein the first reagents comprise fluorescein-labeled free triiodothyronine resistance antibodies, buffer solutions with potential of hydrogen(pH) 7-9, and the concentration of the fluorescein-labeled free triiodothyronine resistance antibodies is 0.5mug / mL-1mug / mL; the second reagents comprise an alkaline-phosphatase-labeled free triiodothyronine antigens, buffer solutions with pH 7-9, and the concentration of the alkaline-phosphatase-labeled free triiodothyronine antigens is 0.02 mug / mL-0.1mug / mL; the magnetic separation reagents are suspension liquid of magnetic particles covered with fluorescein-resistance antibodies. The free triiodothyronine nanometer magnetic particle chemiluminescence assay kit and the preparation method thereof and the detection method thereof have the advantage of enabling the free triiodothyronine 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 chemiluminescence immunoassay method capable of accurately, sensitively, rapidly and quantitatively detecting free triiodothyronine (FT3) in human serum samples, and a kit and a kit used in the method The preparation method. Background technique [0002] Triiodothyronine (3,5,3'-Triiodothyronine, T3) is an iodinated tyrosine with a molecular weight of 651, which is an important thyroid hormone like thyroxine (T4) . It is used to maintain thyroid function and participate in the metabolism of the three major nutrients of sugar, lipids and protein. Triiodothyronine is one of the first choice indicators for hyperthyroidism and can be used as a reliable indicator for judging the efficacy. 99.7% of triiodothyronine exists in the human body in the form of binding to protein, and the content of free triiodothyronine (FT3) that really exerts physiological effects is very low. [000...

Claims

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Application Information

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