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Fluorescence immunosorbent assay kit for joint detection of SAA (Serum Amyloid A) and CRP (C-reactive protein) based on two-color quantum dots and preparation method of fluorescence immunosorbent assay kit

A detection kit and fluorescence immunology technology, applied in biological testing, material inspection products, etc., can solve the problems of further improvement of stability and sensitivity, and high detection cost of chemiluminescence detection kits, so as to reduce technical difficulty and operational errors, Stable and accurate detection results and stable fluorescence intensity

Inactive Publication Date: 2019-04-23
HENAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In most tertiary hospitals, chemiluminescence has basically replaced ELISA as the mainstream, but the detection cost of chemiluminescence detection kits is high, and the stability and sensitivity need to be further improved

Method used

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  • Fluorescence immunosorbent assay kit for joint detection of SAA (Serum Amyloid A) and CRP (C-reactive protein) based on two-color quantum dots and preparation method of fluorescence immunosorbent assay kit
  • Fluorescence immunosorbent assay kit for joint detection of SAA (Serum Amyloid A) and CRP (C-reactive protein) based on two-color quantum dots and preparation method of fluorescence immunosorbent assay kit
  • Fluorescence immunosorbent assay kit for joint detection of SAA (Serum Amyloid A) and CRP (C-reactive protein) based on two-color quantum dots and preparation method of fluorescence immunosorbent assay kit

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preparation example Construction

[0043] In the present invention, the polymer encapsulation method is preferably described by taking polymaleic anhydride cetyl ester modified CdSe / ZnS quantum dots as an example. The preparation method of described poly-cetyl maleic anhydride modified CdSe / ZnS quantum dots, preferably comprises the following steps:

[0044] ①CdSe / ZnS quantum dots are mixed with chloroform and ultrasonicated to obtain a dispersion;

[0045] ② polycetyl maleic anhydride and chloroform are mixed, and ultrasonicated to obtain a polycetyl maleic anhydride chloroform solution;

[0046] ③ mixing the dispersion with the polycetyl maleic anhydride chloroform solution, ultrasonically removing the chloroform to obtain the poly cetyl maleic anhydride-CdSe / ZnS conjugate;

[0047] ④ Mix the polycetyl maleic anhydride-CdSe / ZnS conjugate, water and ammonia water, stir, and separate the solid and liquid to obtain the polycetyl maleic anhydride modified CdSe / ZnS quantum dots.

[0048] In the present invention...

Embodiment 1

[0081] Composition and preparation of the kit

[0082] (1) Composition of the kit

[0083] Coating antibody 1: CRP monoclonal antibody, purchased from Beijing Baichuan Feihong Biotechnology Co., Ltd., the product catalog number is 7D9, and the concentration is 2.2 mg / mL. SAA monoclonal well antibody was purchased from Shanghai Unionwell Biotechnology Co., Ltd., the product catalog number is 2201, and the concentration is 5.0 mg / mL.

[0084] Conjugated antibody 2: CRP monoclonal antibody, purchased from Hangzhou Qitai Co., Ltd., the product catalog number is MC02, and the concentration is 5 mg / mL. SAA monoclonal well antibody was purchased from Shanghai Unionwell Biotechnology Co., Ltd., the product catalog number is 2203, and the concentration is 5.1 mg / mL.

[0085] Fluorescence-labeled antibody 2: green quantum dot-bound CRP monoclonal well antibody, red quantum dot-bound SAA monoclonal well antibody.

[0086] Standard products: CRP standard products were purchased from Ha...

Embodiment 2

[0099] Detection method

[0100] (1) Preparation of standard curve

[0101] (1) Incubation of standard products: Take out the microplate and return to room temperature. Dilute the CRP and SAA standards with sample diluent to the target concentration, for example, the concentration of CRP standard solution: 0, 5, 10, 25, 50, 100, 200, 400, 800, 1000ng / mL; the concentration of SAA standard Solution: 0, 5, 10, 25, 50, 100, 200, 400, 800, 1000 ng / mL. After mixing, add 100 μL to each well, place in a constant temperature and humidity incubator, and incubate at 37°C for 30 minutes; shake off the reaction solution, wash with washing solution five times, and pat dry on absorbent paper.

[0102] (2) Incubation of labeled antibody: dilute SAA antibody 2 with antibody 2 diluent, dilute 1:100 times, 100 μL per well, put it in a constant temperature and humidity incubator, and incubate at 37°C for 30 minutes. Shake off the reaction solution, wash five times with washing solution, and pa...

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Abstract

The invention provides a fluorescence immunosorbent assay kit for joint detection of SAA (Serum Amyloid A) and CRP (C-reactive protein) based on two-color quantum dots and a preparation method of thefluorescence immunosorbent assay kit, and belongs to the technical field of in vitro diagnostic detection. According to the fluorescence immunosorbent assay kit, the advantages of quantum dot multicolor marking and the characteristics of antibody antigen specific reaction are fully utilized; and two antibodies are coated onto the same enzyme coated plate hole to form two kinds of double antibody sandwich methods of coated antibody-to-be-tested antigen-quantum dot labeled antibodies, and a novel simple, sensitive and stable detection method of various markers is established. Compared with a traditional ELISA (Enzyme-linked immunosorbent assay) method, the method has the advantages that the use amount of detection samples is reduced and the detection time is shortened; in addition, the detection efficiency can be improved; the detection cost is favorably saved; and the medical cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnosis and detection, specifically a fluorescent immunoadsorption detection kit based on two-color quantum dots for joint detection of SAA and CRP and a preparation method thereof. Background technique [0002] C-reactive protein (CRP) is an acute phase protein synthesized by hepatocytes. Under normal circumstances, CRP exists at a low level in serum, but in the event of inflammation, infection, or tissue damage, CRP begins to increase within a few hours under the stimulation of cytokines, etc., and reaches the peak within 48 hours. Increased by 1000 times. CRP levels are also closely related to malignant tumors, active phases of autoimmune diseases (rheumatic fever, systemic lupus erythematosus, rheumatoid, etc.), diabetes, and neonatal sepsis. Therefore, the detection of CRP as an important marker of inflammation is widely used in clinical practice. At the same time, studies have found th...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 吴瑞丽吕雁冰李林松申怀彬李金洁
Owner HENAN UNIVERSITY
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