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Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit

A technology of chorionic gonadotropin and nano-magnetic particles, which is applied in the direction of chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, which can solve problems affecting detection sensitivity and accuracy, and achieve good accuracy , precision testing, and low production costs

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

AI Technical Summary

Problems solved by technology

[0007] However, in the actual immunoassay, due to the large amount of impurities contained in the sample to be tested, which affects the detection sensitivity and accuracy to a certain extent, the target analyte can be quickly separated and purified from the complex sample matrix. It is one of the problems faced by clinical laboratory workers

Method used

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  • Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit
  • Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit
  • Nano magnetic particle chemiluminiscence assay kit for free HCG (human chorionic gonadotropin) beta subunit, preparation method for Nano magnetic particle chemiluminiscence assay kit and detection method adopting Nano magnetic particle chemiluminiscence assay kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the preparation of magnetic separation reagent.

[0049] Materials and Instruments

[0050] Suspension of magnetic particles: the content of magnetic particles is 5wt%, containing carboxyl (COOH) active groups, and the carboxyl content per gram (g) of magnetic particles (dry weight) is not less than 0.4 millimoles (mmol), with superparamagnetism, diameter Between 0.5-2μm.

[0051] Anti-FITC antibody: it can be a polyclonal antibody or a monoclonal antibody, the purity should exceed 90wt%, and the diluted titer should exceed 1:1 million.

[0052] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0053] Steps

[0054] 1. Take 100mg suspension of magnetic particles, remove the supernatant by magnetic separation, and resuspend in 10ml of 0.05mol / L, PH4.5-5MES buffer;

[0055] 2. Add 2-4mg of anti-FITC antibody and suspend at room temperature for 30-60min;

[0056] 3. Add 0.5-1ml, freshly pr...

Embodiment 2

[0058] Embodiment 2, the preparation of the first reagent.

[0059] Materials and Instruments

[0060] Anti-F-β-HCG monoclonal antibody, prepared by Beijing Leadman Biochemical Co., Ltd., with a purity of more than 95wt%, a concentration of 2mg / ml, and stored in phosphate buffer;

[0061] Fluorescein isothiocyanate (FITC), sodium carbonate and other reagents should be chemically pure,

[0062] G-25 gel purification column was purchased from GE Company.

[0063] Steps

[0064] 1. Prepare 0.5mg / ml FITC solution with 0.1-0.2mol / L carbonate buffer solution with pH9.0-10.0;

[0065] 2. According to the molar ratio of anti-F-β-HCG antibody to FITC molecule 1:20, add FITC solution to the antibody solution, mix well, and let it stand at room temperature for more than 12 hours to generate anti-F-β-HCG antibody-FITC conjugate ;

[0066] 3. Separate the reaction liquid after step 2 with G-25 gel column, remove unreacted FITC, and obtain anti-F-β-HCG antibody-FITC conjugate (ie, FITC...

Embodiment 3

[0067] Embodiment 3, preparation of the second reagent.

[0068] Materials and Instruments

[0069] Anti-F-β-HCG monoclonal antibody, prepared by Beijing Leadman Biochemical Co., Ltd., with a purity of more than 95%, a concentration of more than 1mg / ml, and stored in phosphate buffer;

[0070] The purity of alkaline phosphatase is about 99%, the specific activity is about 1500U / mg, and the concentration is 10mg / ml;

[0071] Coupling agent succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 2-iminothiophene (2-IT) were purchased from THERMO company , Chemical reagents such as TRIS should be chemically pure;

[0072] AKTA-purifier protein purification instrument and Supperde x200 gel purification column are products of GE Company.

[0073] Steps

[0074] 1. Take 1mg of anti-F-β-HCG antibody, add 2-4μl of 10mg / ml coupling agent 2-IT solution, let stand at room temperature for 20min, add 10μl of 0.1mol / L glycine solution, and let stand at room temperature for 5...

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Abstract

The invention relates to a nano magnetic particle chemiluminiscence assay kit for a free HCG (human chorionic gonadotropin) beta subunit, a preparation method for the Nano magnetic particle chemiluminiscence assay kit and a detection method adopting the Nano magnetic particle chemiluminiscence assay kit. The kit comprises a first agent, a second agent and a magnetic separation agent, wherein the first agent comprises solution of free HCG beta subunit antibody marked with fluorescein isothiocyanate; the second agent comprises solution of free HCG beta subunit antibody marked with alkaline phosphatase; and the magnetic separation agent comprises suspension of magnetic particles coated with a fluorescein isothiocyanate resistance antibody. The invention further discloses the preparation method of the kit and the detection method for detecting the HCG beta subunit by adopting the kit. Compared with the prior art, the nano magnetic particle chemiluminiscence assay kit has better accuracy, higher precision and higher sensitivity; a sample to be detected does not need to be pre-diluted; and the nano magnetic particle chemiluminiscence assay kit is simple and time-saving to operate, has a wide detection range and is low in cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection method for free human chorionic gonadotropin beta subunit, a detection kit and a preparation method of the kit. Background technique [0002] Human chorionic gonadotropin (HCG) is a glycoprotein produced by the syncytiotrophoblast of the placental chorion, which is composed of α and β units bound together by ionic and hydrophobic bonds. The α subunit is shared by the anterior pituitary hormones, and it is similar in structure to the α subunits of FSH, LH, and TSH. The structure of the β subunit is different from the β subunit of LH, etc., and can be distinguished in terms of immunological activity. Most of the β subunits in the blood exist in the form of whole molecules, and the free β subunits (F-β-HCG) account for about 1% to 5% of HCG. The free β subunit in the blood is produced 6 to 8 days after conception, reaches a peak at 50 to 80 days, and then declines steadily...

Claims

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Application Information

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