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Bacterial endotoxin detecting test paper and kit

A technology for bacterial endotoxin detection and test paper, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of sample and specimen preparation to be tested, affecting the measurement results, poor specificity, etc., and achieves low detection cost, convenient detection, and low cost. low effect

Inactive Publication Date: 2019-08-06
WUXI PEOPLES HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The method for detecting endotoxin based on Limulus reagent has the advantages of rapidity, simplicity, high sensitivity and easy promotion. It has always been the standard method for detecting endotoxin in the world, but it still has the following disadvantages: (1) poor specificity
(2) Preparation of samples and specimens to be tested
Therefore, some components of the sample to be tested may interfere with a certain step of the cascade reaction and affect the final determination result.
In addition, the lipid components of endotoxin may be hidden inside the unit when they are aggregated in aqueous solution, and cannot fully contact with LAL to affect the measurement results.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Synthesis of Polypeptides

[0033] According to the following amino acid sequence, the limulus factor C polypeptide is chemically synthesized, and its amino acid sequence is as follows:

[0034] GFKLKGMARISCLPNGQWSNFPPKCIRECAMVSSHAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLM.

[0035] Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize; purity > 90%.

Embodiment 2

[0036] Example 2 Fluorescence-labeled Limulus Factor C Polypeptide

[0037] Cleaning: Take fluorescent microspheres (purchased from Bangs Lab, product number: 11233) into a centrifuge tube, add 0.1M MES (2-morpholinoethanesulfonic acid) (pH 5.0) buffer and mix well, and run at 13000rpm for 15min, Centrifuge at 4°C, discard the supernatant, and resuspend in 0.1M MES (pH 5.0) buffer for use. Activation and cleaning: the activator EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and fluorescent microspheres according to the mass The ratio is 2:1.5:2 for activation, the specific operation is as follows:

[0038] Weigh EDC and NHS and add them to 0.1M MES (pH 5.0) buffer solution to dissolve, quickly take an appropriate amount into the washed fluorescent microspheres, seal them with a parafilm and place them on a 200rpm shaker at room temperature for 30min, take them out at 13000rpm for 30min , centrifuged at 4°C to remove the supern...

Embodiment 3

[0044] Example 3 Fluorescently labeled anti-bacterial endotoxin antibody

[0045] Cleaning: Take fluorescent microspheres (purchased from Bangs Lab, product number: 11233) into a centrifuge tube, add 0.1M MES (pH5.0) buffer, mix well, and centrifuge at 13000rpm, 15min, 4°C, discard the supernatant, Resuspend with 0.1M MES (pH 5.0) buffer for use. Activation and cleaning: Activate the activator EDC, NHS and fluorescent microspheres according to the mass ratio of 2:1.2:2, the specific operation is as follows:

[0046] Weigh EDC and NHS and add them to 0.1M MES (pH 5.0) buffer solution to dissolve, quickly take an appropriate amount into the washed fluorescent microspheres, seal them with a parafilm and place them on a 200rpm shaker at room temperature for 30min, take them out at 13000rpm for 30min , centrifuged at 4°C to remove the supernatant, resuspended with an equal amount of 0.1M MES (pH 6.5) buffer solution, ultrasonically mixed and washed, repeating the above operation...

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Abstract

The invention provides bacterial endotoxin detecting test paper. The test paper comprises a cellulose membrane and a conjugate pad, a bacterial endotoxin antibody is attached onto the cellulose membrane, limulus C factor polypeptide by gene engineering recombination wraps the conjugate pad, and the bacterial endotoxin antibody, limulus C factor polypeptide by gene engineering recombination and bacterial endotoxin make specific combination to form a sandwich structure. The disclosed bacterial endotoxin detecting test paper and kit are independent from natural animal serum and not limited by rawmaterials, the cost can be reduced, the detection process of the test paper and kit is highly automatic, and the test paper and kit are suitable for large-scale production.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a rapid detection test paper and a detection kit for endotoxin. Background technique [0002] Bacterial endotoxin is produced by Gram-negative bacteria and is part of the outermost structure of its cell wall, lipopolysaccharide (LPS), which consists of three parts: polysaccharide O antigen, core polysaccharide and lipid A, of which lipid A is endotoxin The main group of various biological activities or toxic reactions. Bacterial endotoxins are only released when the bacteria die, disintegrate or artificially destroy the bacterial structure, and can cause pathogenic effects on the body. The human body is extremely sensitive to endotoxin, and a very small amount (1-5ng / kg body weight) of endotoxin can raise the body temperature, produce hypotension and shock, disseminated intravascular coagulation, insufficient blood supply and hypoxia lead to metabolic symptoms and o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558
CPCG01N33/558
Inventor 王春新徐新平赵静
Owner WUXI PEOPLES HOSPITAL
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