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

A technology of triiodothyronine and protosine, which is applied in chemiluminescence/bioluminescence, biological testing, and analysis by chemical reaction of materials, etc. Control stability and other issues, to achieve the effect of low production cost, good accuracy, and small difference between analysis batches

Active Publication Date: 2015-03-11
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

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Experimental program
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Embodiment 1

[0041] Embodiment 1 The preparation of the first reagent

[0042] (1) Materials and instruments: anti-FT3 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.

[0043] (2) Preparation steps:

[0044] ① Use 0.1~0.2mol / L carbonate buffer solution with pH9.0~10.0 to prepare 0.5mg / mL FITC solution;

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

[0046] ③Separate the reaction liquid after step ② through G-25 gel column, remove unreacted FITC, and obtain a solution containing FT3 antibody-FITC conjugate (ie, FITC-labeled FT3 antibody);

[0047] ④Dilute the solution containing FT3 antibody-...

Embodiment 2

[0048] Embodiment 2 Preparation of the second reagent

[0049] (1) Materials and instruments: FT3 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.

[0050] (2) Preparation steps:

[0051] ① Take 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 it at 2-8 °C for later use;

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

Embodiment 3

[0054] The preparation of embodiment 3 magnetic separation reagents

[0055] (1) Materials and instruments:

[0056] 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;

[0057] 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;

[0058] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0059] (2) Preparation steps:

[0060] 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;

[0061] ②Add 2~4mg of anti-FITC antibody and suspend at room tem...

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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 in particular relates to a chemiluminescence immunoassay method capable of accurately, sensitively, rapidly and quantitatively detecting free triiodothyronine (FT3) in human serum samples, a kit and a kit used in the method method of preparation. Background technique [0002] Triiodothyronine (3,5,3'-triiodothyronine, T3), an iodotyrosine with a molecular weight of 651, is an important thyroid hormone like thyroxine (T4) . It is used to maintain thyroid function and participate in the metabolic physiological functions of the three major nutrients of sugar, lipid and protein. Triiodothyronine is one of the preferred indicators of hyperthyroidism and can be used as a reliable indicator for judging the curative effect. 99.7% of triiodothyronine exists in the form of protein binding in the human body, while the content of free triiodothyronine (FT3) that actually exerts physiological effects is ve...

Claims

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Application Information

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Patent Type & Authority Patents(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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