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Endotoxin latex microspheres as well as preparation method and application thereof

An endotoxin and microsphere technology, applied in the preparation of microspheres, microcapsule preparations, instruments, etc., can solve the problems of large deviation of experimental results, large interference, and inability to use, achieve good stability, enhance scattering signals, and improve detection. The effect of efficiency

Active Publication Date: 2022-05-31
JAFRON BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, bovine serum albumin is often used as a blocking reagent, but since there is endotoxin that cannot be removed in bovine serum albumin, the interference caused by using it as a blocking reagent for endotoxin detection reagents will be large, and the deviation of the experimental results is large. Therefore, it cannot be used in the preparation of endotoxin detection reagents

Method used

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  • Endotoxin latex microspheres as well as preparation method and application thereof
  • Endotoxin latex microspheres as well as preparation method and application thereof
  • Endotoxin latex microspheres as well as preparation method and application thereof

Examples

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preparation example Construction

[0031] combine figure 1 As shown, the first aspect of the embodiment of the present application provides a method for preparing endotoxin latex microspheres, comprising the following steps:

[0032] Step S1, mixing and reacting latex microspheres with carboxyl groups and ethyldimethylaminopropyl carbodiimide, after the reaction is completed, adding hydrazine hydrate to continue the mixing reaction to obtain activated latex microspheres;

[0033] Step S2, using sodium periodate to oxidize the hydroxyl group at the Fc end of the non-binding active region of the endotoxin into an aldehyde group;

[0034] Step S3, mixing the activated latex microspheres with the aldehydelated endotoxin, and adding aldehydelated polyethylene glycol to seal the unreacted groups on the latex microspheres after the reaction, and then adding Sodium borohydride continues to react to obtain latex microspheres coupled with endotoxin.

[0035] In the examples of the present application, the Fc end of the...

Embodiment 1

[0061] This embodiment provides a method for preparing endotoxin latex microspheres, comprising the following steps:

[0062] (1) Take 0.1 mg of carboxylated latex microspheres with a particle size of 130-200 nm (commercialized carboxylated latex microspheres from Bangs Lab Company), wash twice with 10-50 mmol / LMES buffer (pH 4.0); add Add a certain volume of buffer solution, add 0.1mg of EDC (purchased by sigma company), shake and react at room temperature for 15-30min, after the reaction, centrifuge to remove the supernatant, wash with buffer solution twice, add excess hydrazine hydrate (N 2 h 4 ) solution, so that the final concentration of the carboxylated latex microspheres in the mixed solution is about 0.1% w / v, shake and react at normal temperature for 90-120min, after the reaction finishes, centrifuge to remove the supernatant, and use 10-50mmol / L boron Salt buffer (pH is 8.0) was washed twice to obtain activated latex microspheres;

[0063] (2) Take 0.5uL 5mg / mL en...

Embodiment 2

[0066] This example provides a method for verifying the non-specific recognition of endotoxin latex microspheres, prepared respectively according to the preparation method in Example 1:

[0067] (a) Bare spheres: carboxylated latex microspheres;

[0068] (b) EDC-activated latex microspheres: refer to the latex microspheres only activated with EDC in the embodiment 1 step (1);

[0069] (c) grafted hydrazine hydrate latex microspheres after activation: refer to the activated latex microspheres prepared in Example 1 step (1);

[0070] (d) Latex microspheres grafted with hydrazine hydrate and endotoxin after activation: refers to the reaction product after the mixed reaction between the aldylated endotoxin and the activated latex microspheres at room temperature in step (3) of Example 1;

[0071] (e) Latex microspheres grafted with hydrazine hydrate and endotoxin after activation and blocked with PEG-550: refer to the coupled endotoxin latex microspheres blocked with aldehydelate...

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Abstract

The invention provides endotoxin latex microspheres and a preparation method and application thereof.The preparation method of the endotoxin latex microspheres comprises the steps that latex microspheres with carboxyl and ethyl dimethyl amine propyl carbodiimide are mixed for reaction, after the reaction is finished, hydrazine hydrate is added for continuous mixing reaction, and activated latex microspheres are obtained; oxidizing hydroxyl at the Fc terminal of the non-binding active region of the endotoxin into an aldehyde group by using sodium periodate; mixing the activated latex microspheres with aldehyde endotoxin to react, adding aldehyde polyethylene glycol to seal unreacted groups on the latex microspheres after the reaction is finished, and adding sodium borohydride to continue the reaction after the reaction is finished, so as to obtain the latex microspheres coupled with the endotoxin. Interference of endotoxin in the environment on a sample detection result can be eliminated, and the accuracy and credibility of the detection result can be improved.

Description

technical field [0001] The invention relates to the technical field of bacterial endotoxin detection, in particular to an endotoxin latex microsphere and a preparation method and application thereof. Background technique [0002] Endotoxin is the main component of the outer wall of the cell membrane of gram-negative bacteria (GNB), and plays an important role in the stability of bacteria. Bacterial endotoxin molecule is an amide lipid complex with a disaccharide structure, and its molecular weight is generally between 2000 and 2300. Because of its small molecular weight, it is very easy to invade from the mesentery or interstitial fluid, and enter the circulatory system with the blood. Endotoxin is almost ubiquitous, is a highly toxic inflammatory and pyrogenic substance, and is the main pathogenic medium of endotoxemia and septic shock. Endotoxemia is a condition caused by the release of a large amount of bacterial endotoxin into the blood by bacteria in the blood or in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J13/02G01N33/543G01N33/532
CPCB01J13/02G01N33/54353G01N33/532Y02A50/30
Inventor 董凡金宗文陈鹏
Owner JAFRON BIOMEDICAL
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