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Nano magnetic particle chemiluminiscence determining kit for promoting hormone generation by follicles and preparation and detection method thereof

A follicle-stimulating, nano-magnetic particle technology, applied in the biological field, can solve problems affecting detection sensitivity and accuracy, and achieve cost advantages, low production costs, and precise detection effects

Active 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

[0006] 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 determining kit for promoting hormone generation by follicles and preparation and detection method thereof
  • Nano magnetic particle chemiluminiscence determining kit for promoting hormone generation by follicles and preparation and detection method thereof
  • Nano magnetic particle chemiluminiscence determining kit for promoting hormone generation by follicles and preparation and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Preparation of magnetic separation reagent.

[0043] Materials and Instruments

[0044] 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.

[0045] Anti-FITC antibody: it can be polyclonal antibody or monoclonal antibody, the purity should be more than 90%, and the dilution titer should be more than 1:1 million.

[0046] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0047] Steps

[0048] 1. Take 100 mg of magnetic particle suspension, magnetically separate to remove the supernatant, and resuspend in 10 ml of 0.05mol / L, pH4.5-5 MES buffer;

[0049] 2. Add 2-4mg of anti-FITC antibody and suspend at room temperature for 30-60min;

[0050] 3. Add 0.5-1ml, fres...

Embodiment 2

[0052] Example 2 Preparation of the first reagent.

[0053] Materials and Instruments

[0054] Anti-FSH monoclonal antibody, with a purity of more than 95% by weight and a concentration of more than 2 mg / ml, stored in phosphate buffer;

[0055] Fluorescein isothiocyanate (FITC), sodium carbonate and other reagents should be chemically pure,

[0056] G-25 gel purification column was purchased from GE Company.

[0057] Steps

[0058] 1. Prepare 0.5mg / ml FITC solution with 0.1-0.2mol / L carbonate buffer solution at pH9.0-10.0;

[0059] 2. According to the molar ratio of anti-FSH antibody and 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;

[0060] 3. Use a G-25 gel column to separate the antibody FITC conjugate from free FITC to obtain a concentrated antibody solution coupled with FITC molecules, and use the solution to contain 0.5% bovine serum albumin (BSA) at pH 8.0 The 0.1mol / L TRIS buf...

Embodiment 3

[0061] Example 3 Preparation of the second reagent.

[0062] Materials and Instruments

[0063] Anti-FSH monoclonal antibody, with a purity of more than 95%, a concentration of more than 1mg / ml, stored in phosphate buffer;

[0064] The purity of alkaline phosphatase is about 99%, the specific activity is about 1500U / mg, and the concentration is 10mg / ml;

[0065] 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;

[0066] AKTA-purifier protein purification instrument and Supperdex200 gel purification column are products of GE Company.

[0067] Steps

[0068] 1. Take 1mg of anti-FSH 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, let stand at room temperature for 5min, gel with G-25 Desalt the column, collect the activated antibody, a...

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Abstract

The invention relates to a nano magnetic particle chemiluminiscence determining kit for promoting hormone generation by follicles and preparation and detection method thereof. The kit comprises a first reagent which comprises a follicle-stimulating hormone antibody solution containing fluorescein isothiocyanate, a second reagent which comprises a follicle-stimulating hormone antibody solution containing an alkaline phosphatase mark, and a magnetic separation reagent which comprises a magnetic particle suspension coated by a fluorescein isothiocyanate antibody. The invention further discloses a preparation method of the kit and a detection method for detecting generated hormones by promoting the follicles by using the kit. Compared with the prior art, the kit is better in accuracy, higher in precision and higher in sensitivity, and samples to be tested are not needed to be pre-diluted, so that the kit is simple and time-saving to operate, wide in detection range and low in cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nano-magnetic particle chemiluminescence assay kit for follicle-stimulating hormone, a preparation method and a detection method thereof. Background technique [0002] Follicle Stimulating Hormone (FSH) is a glycoprotein hormone secreted by the anterior pituitary gland. Its molecular weight is about 30,000 and it consists of two polypeptide chains (that is, α and β subunits). The α subunit of FSH consists of 92 It is composed of three amino acid residues and has the same structure as the α subunit of LH, TSH and hCG, and the β subunit is different among the above hormones, thus giving each hormone its own biological and immunological characteristics. Its main function is to promote the development and maturation of ovarian follicles, mainly to promote the proliferation and differentiation of follicular granulosa cells, and to promote the growth of the entire ovary. When it acts on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/533
Inventor 于大为程晓蕾
Owner SUZHOU HAOOUBO BIOPHARML
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