Kit for detecting nano magnetic particle chemiluminescence of hormothyrin and preparation method and detection method thereof

A technology of thyroid-stimulating hormone 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 and use costs, limited detection method precision, antibody Complicated preparation and other problems, to achieve the effect of low production cost, good accuracy, simple operation and time-saving

Active Publication Date: 2015-07-01
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of preparation and use of the kit is high because, on the one hand, when preparing the magnetic particle suspension coated with anti-thyroid-stimulating hormone monoclonal antibody, not only the process is complicated, but also the anti-thyroid-stimulating hormone monoclonal antibody is directly The coating rate of thyroid hormone monoclonal antibody coated on magnetic particles is low, resulting in higher cost; on the other hand, it uses horseradish peroxidase-labeled anti-thyroid stimulating hormone monoclonal antibody, the preparation of the antibody It is also very cumbersome, and the labeling rate is low, which limits its detection effect and increases the cost
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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  • Kit for detecting nano magnetic particle chemiluminescence of hormothyrin and preparation method and detection method thereof
  • Kit for detecting nano magnetic particle chemiluminescence of hormothyrin and preparation method and detection method thereof
  • Kit for detecting nano magnetic particle chemiluminescence of hormothyrin and preparation method and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 The preparation of the first reagent

[0046] (1) Materials and instruments: Thyroid-stimulating hormone (TSH) monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC), sodium carbonate and other reagents It should be chemically pure; G-25 gel purification column was purchased from GE.

[0047] (2) Preparation steps:

[0048] ① Prepare 0.5mg / mL FITC solution with 0.1~0.2mol / L carbonate buffer solution with pH 9.0~10.0;

[0049] ② Add the FITC solution prepared in step ① to the antibody solution according to the ratio of TSH antibody to FITC molecular ratio of 1:20, mix well, and let stand at room temperature for 12 hours to generate TSH antibody-FITC conjugate;

[0050] ③Separate the reaction liquid after step ② through G-25 gel column, remove unreacted FITC, and obtain a solution containing TSH antibody-FITC conjugate (i.e. FITC-labeled TSH antibody);

[0051] ④ Dilute the solution co...

Embodiment 2

[0052] Embodiment 2 Preparation of the second reagent

[0053] (1) Materials and instruments: TSH monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2 mg / mL); alkaline phosphatase preserved in phosphate buffer (ALP solution, ALP purity of about 99 %, the specific activity is about 1500U / mg, and the concentration is 10mg / mL); the cross-linking agent SMCC, 2-IT are purchased from THERMO company, TRIS and other chemical reagents should be chemically pure; G-25 gel purification column is a product of GE company .

[0054] (2) Preparation steps:

[0055] ①Take 1mg of TSH antibody, add 2-4μL of 10mg / mL coupling agent 2-IT solution, let it stand at room temperature for 20min, add 10μL of 0.1mol / L glycine solution, let it stand at room temperature for 5min, remove it with G-25 gel column salt, collect the activated TSH antibody, and store it at 2-8°C for later use;

[0056] ② Take 1.5 mg of ALP solution, add 10-20 μL of 5 mg / mL SMCC solution, let it...

Embodiment 3

[0059] The preparation of embodiment 3 magnetic separation reagents

[0060] (1) Materials and instruments:

[0061] 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.

[0062] 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;

[0063] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0064] (2) Preparation steps:

[0065] 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;

[0066] ②Add 2~4mg of anti-FITC antibody and suspend at room te...

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Abstract

The invention relates to a kit for detecting nano magnetic particle chemiluminescence of hormothyrin and a preparation method and a detection method thereof. The kit comprises a hormothyrin antibody solution containing a fluorescein mark, a magnetic particle suspension liquid coated by a magnetic particle antibody, and a hormothyrin antibody solution containing an alkaline phosphatase mark. According to the invention, hormothyrin can be quantitively detected 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 thyroid-stimulating hormone (TSH) nanometer magnetic particle chemiluminescence assay kit combined with immunomagnetic particle separation technology and chemiluminescence immunoassay technology, as well as a preparation method and a detection method thereof. Background technique [0002] Thyroid stimulating hormone (TSH) is a glycoprotein secreted by the pituitary gland with a molecular weight of about 28,000. It consists of two subunits, α and β. The alpha subunit of TSH interacts with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secreted by the pituitary gland, and human chorionic gonadotropin (HCG) produced by the placenta )basically the same. Their difference is mainly in the amino acid sequence of the β subunit, which makes these hormones have different biological characteristics, and they can be distinguished by immunoassay methods. [0003]...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/74G01N21/76G01N33/532
Inventor 于大为程晓蕾赵文姬
Owner SUZHOU HAOOUBO BIOPHARML
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