A fluorescent microsphere for labeling specific high-affinity recombinant antibody and its application

A fluorescent microsphere and specific technology, applied in the field of biomedicine, can solve the requirements that immunofluorescence detection reagents are difficult to achieve high sensitivity and high precision, immunofluorescence microparticles have low life activity, and fluorescent microparticle labeling efficiency. Low problems, to achieve accurate and reliable quantitative results, improve reagent sensitivity and linear range, easy and fast operation

Active Publication Date: 2019-06-21
江苏博华医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, traditional methods or mechanical centrifugation methods in other patents purify the fluorescent particles during the labeling process. This method has low labeling efficiency of fluorescent particles and low activity of immunofluorescent particles.
[0012] However, the content of the above-mentioned specific biomarkers in human blood or urine is low, usually in pg to ng / mL, and it is difficult for the immunofluorescence detection reagents prepared by traditional methods to achieve the required high sensitivity and high precision requirements

Method used

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  • A fluorescent microsphere for labeling specific high-affinity recombinant antibody and its application
  • A fluorescent microsphere for labeling specific high-affinity recombinant antibody and its application
  • A fluorescent microsphere for labeling specific high-affinity recombinant antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] In Example 1 and Comparative Examples 1 and 2:

[0072] Fluorescent microsphere liquid: particle size of 300nm, solid content of 1%;

[0073] EDC solution: 50mg / ml 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide (EDC);

[0074] NHS solution: 50mg / ml N-hydroxysuccinimide (NHS);

[0075] MES buffer: 0.1M MES PH 5N-morpholine ethanesulfonic acid (MES);

[0076] PBS buffer: 0.05M PBS PH 7.4 phosphate (PBS);

[0077] Amino polyethylene glycol carboxyl (NH 2 -PEG-COOH), the molecular weight is 2000 Dalton.

[0078] Fluorescent microsphere compound solution: containing 0.2wt% BSA, 0.1wt% Trilx X-100, 0.08wt% sodium azide, 0.1M, PH8.0 phosphate buffer.

[0079] Preparation of fluorescence detection card for rapid quantitative detection of KIM-1 in human urine

[0080] 1. Preparation of KIM-1 antibody labeled fluorescent microspheres:

[0081] 1.1 Take 1ml of fluorescent microsphere solution, add 0.1ml of EDC solution, and activate at room temperature for 15 minutes;

[0082] 1.2 Add 0.1ml of NHS...

Embodiment 2

[0181] In Example 2 and Comparative Examples 3 and 4:

[0182] Fluorescent microsphere liquid: particle size of 300nm, solid content of 1%;

[0183] EDC solution: 50mg / ml 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide (EDC);

[0184] NHS solution: 50mg / ml N-hydroxysuccinimide (NHS);

[0185] MES buffer: 0.1M MES PH 5N-morpholine ethanesulfonic acid (MES);

[0186] PBS buffer: 0.05M PBS PH 7.4 phosphate (PBS);

[0187] Amino polyethylene glycol carboxyl (NH 2 -PEG-COOH), the molecular weight is 2000 Dalton.

[0188] Fluorescent microsphere compound solution: containing 0.2wt% BSA, 0.1wt% Trilx X-100, 0.08wt% sodium azide, 0.1M, PH8.0 phosphate buffer.

[0189] Preparation of fluorescence detection card for rapid quantitative detection of NGAL in blood

[0190] 1. Preparation of NGAL antibody labeled fluorescent microspheres:

[0191] 1.1 Take 1ml of fluorescent microsphere solution, add 0.1ml of EDC solution, and activate at room temperature for 15 minutes;

[0192] 1.2 Add 0.1ml of NHS solutio...

Embodiment 3

[0293] In Example 3 and Comparative Examples 5 and 6:

[0294] Fluorescent microsphere liquid: particle size 250nm, solid content 1%;

[0295] EDC solution: 50mg / ml 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide (EDC);

[0296] NHS solution: 50mg / ml N-hydroxysuccinimide (NHS);

[0297] MES buffer: 0.1M MES PH 5N-morpholine ethanesulfonic acid (MES);

[0298] PBS buffer: 0.05M PBS PH 7.4 phosphate (PBS);

[0299] Amino polyethylene glycol carboxyl (NH 2 -PEG-COOH), the molecular weight is 1000 Dalton.

[0300] Fluorescent microsphere compound solution: containing 0.2wt% BSA, 0.1wt% Trilx X-100, 0.08wt% sodium azide, 0.1M, PH8.0 phosphate buffer.

[0301] Preparation of fluorescence detection card for rapid quantitative detection of IGFBP-7 in urine

[0302] 1. Preparation of IGFBP-7 antibody labeled fluorescent microspheres:

[0303] 1.1 Take 1ml of fluorescent microsphere solution, add 0.1ml of EDC solution, and activate at room temperature for 15 minutes;

[0304] 1.2 Add 0.1ml of NHS solutio...

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Abstract

The invention belongs to the field of biological medicine, relates to a specific high-affinity recombinant antibody marking fluorescent microsphere and a preparation method and application thereof and particularly relates to a plug-and-play fluorescence detection card for quick quantitative detection of KIM-1, NGAL, IGFBP7, TIMP-2, IL-18, PCT and cTnI in human urine or blood samples. The method includes: activating fluorescent microspheres; adding the activated fluorescent microspheres into a dialysis bag filled with phosphate buffer to obtain dialyzed fluorescent microsphere solution; adding amino-polyethylene glycol-carboxyl, further activating the fluorescent microsphere solution subjected to secondary dialysis, making with a recombinant antibody, centrifuging after reaction, washing, precipitating, resuspending and precipitating. The fluorescent microsphere has advantages that immunofluorescence reaction lower limit can be remarkably improved to enable sensitivity of a prepared test card to be improved by 10-100 times of that of test cards prepared according to a traditional method, and a test range is remarkably expanded.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a fluorescent microsphere labeled with a specific high-affinity recombinant antibody and a preparation method and application thereof, in particular to a rapid quantitative detection of kidney injury molecules in human urine or blood samples. KIM-1), neutrophil gelatinase-associated apolipoprotein (NGAL), insulin-like growth factor binding protein-7 (IGFBP-7), tissue inhibitor of metalloproteinase-2 (TIMP-2), interleukin-18 (IL -18), Procalcitonin (PCT), Cardiac Troponin I (cTnI) plug-in fluorescence detection card. Background technique [0002] KIM-1 (Kidney Injury Molecule-1) is a transmembrane glycoprotein composed of 334 amino acid residues. It is almost not expressed in normal kidney tissues, but is present in human proximal tubule epithelial cells after ischemia and kidney injury. In the high expression state, its extracellular domain is degraded into soluble fragments und...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/533
CPCG01N33/533G01N33/577G01N33/68
Inventor 王宇飞李强
Owner 江苏博华医药科技有限公司
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