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Preparation and application of chemiluminescent immunoprobe based on dendrimer double-amplification labeling

A chemiluminescent immunity and double amplification technology, which is applied in chemiluminescence/bioluminescence, analysis through chemical reactions of materials, scientific instruments, etc., can solve the problems of not being able to make full use of the amplification modes of biotin and avidin, and being helpless. Achieve the effect of improving the intensity of the luminous signal and enhancing the performance

Inactive Publication Date: 2018-07-31
NANJING TZONE BIOLOGICAL SCI & TECH
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AI Technical Summary

Problems solved by technology

[0004] The general biotin and avidin amplification system adopts an amplification mode, which only utilizes the amplification effect between biotin and avidin or only aims at the increase and amplification of antibody binding amount, and cannot effectively combine biotin and avidin. When the pixel amplification mode is fully utilized, it often seems helpless in the face of some trace detection

Method used

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  • Preparation and application of chemiluminescent immunoprobe based on dendrimer double-amplification labeling

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Experimental program
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Embodiment 1

[0026] Preparation of dual amplification chemiluminescent immunodetection probes:

[0027] 1) Take a certain 10nmol / L dendrimer, add 100μL of 10mg / mL EDC for activation, add 3nmol / L streptavidin, 0.05mol / L carbonate buffer (pH 9.5) to supplement the total volume of 500μL , stirred and reacted at 37°C for 1h;

[0028] 2) After the reaction, add the purchased 0.1mol / L activated signal substance according to the molar ratio of 1:10, stir and react at 37°C for 1h, and finally add 50μL of 0.05mol / L Tris-HCl for blocking reaction and react on a shaking table at 37°C for 30min ;

[0029] 3) After the entire reaction is completed, transfer to a dialysis bag with a molecular weight cut-off of 8KD, the dialysate is 0.1mol / L PBS (pH7.2), dialyze to remove unreacted small molecule chemiluminescent signal substances, and collect the dialyzed solution. Obtaining dendrimers modified with chemiluminescent signal substances and streptavidin for use;

[0030] 4) Take 2.7 μL of the purchased ...

Embodiment 2

[0034] Preparation of magnetic microsphere capture separation probe:

[0035] 1) Take 10 mg of purchased carboxy magnetic microspheres in a centrifuge tube, let it stand on a magnetic separation rack for 1 min, remove the magnetic bead preservation solution, add 1.5mL 0.1mol / L MES buffer, and resuspend the magnetic beads for later use;

[0036] 2) Dissolve the weighed EDC with pre-cooled reaction buffer to 10 mg / mL, take 34 μL and quickly add it to the resuspended magnetic beads, shake and mix immediately (or shake while adding);

[0037] 3) Dissolve the weighed streptavidin in reaction buffer to 10 mg / mL, add 60 μL to the magnetic beads, and shake on a shaker at room temperature (25°C) / 37°C for 3 hours.

[0038]4) Sonicate for 3min-4min, put it on the magnetic separation rack and let it stand for 1min, suck off the reaction solution, wash with Tris-HCl buffer for 3 times, add Tris-HCl buffer containing 2% BSA to resuspend (8mL, 1.25mg / mL), and stored at 2°C to 8°C for later...

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Abstract

The invention relates to a preparation and application of a chemiluminescent immunoprobe based on dendrimer double-amplification labeling, and belongs to the field of chemiluminescent labeling in chemiluminescence in-vitro diagnosis. The preparation method of the amplified chemiluminescence immunodetection probe comprises the following steps: 1, preparation of a dendrimer-streptavidin-chemiluminescent signal substance complex; 2, biotinylation labeling of an antibody; and 3, preparation of a dual-amplification chemiluminescent immunolabeling detection probe complex. According to the dual-amplifying chemiluminescence immunodetection probe, the mode of enhancing the fluorescent intensity and the mode of increasing the detecting molecule combining weight of fluorescent microspheres are effectively combined, one-time amplification is achieved through the combination effect between biotin and avidin, second-time amplification is achieved by use of a signal-substance-labeled dendrimer, and the signal intensity of chemiluminescence immunoassay is greatly improved through the dual amplifying mode.

Description

technical field [0001] The invention belongs to the field of chemiluminescence labeling in chemiluminescence in vitro diagnosis, and in particular relates to a preparation method and application of a double amplification chemiluminescence immunolabeling probe. Background technique [0002] Immunoassay technology is currently one of the most basic and commonly used detection methods in the fields of medicine, biology, food safety and other fields. This technology is based on the principle of specific reaction between antibodies and antigens for qualitative and quantitative analysis of the test object, and is widely used in medical biology. In basic research, clinical medicine, environmental monitoring, disease diagnosis, disease development prediction and even detection of food and microbial contamination. [0003] Chemiluminescence Immunoassay (CLIA) is a combination of high-sensitivity chemiluminescence assay technology and high-specificity immune response for various antig...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/543G01N21/76
CPCG01N21/76G01N33/531G01N33/54326
Inventor 徐林
Owner NANJING TZONE BIOLOGICAL SCI & TECH
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