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High-molecular polymer for stabilizing immobilized protein, and preparation method and application thereof

A complex, bovine serum albumin technology, used in analytical materials, biological testing, material inspection products, etc., can solve the problems of inability to cross-platform application, difficulty in immobilizing proteins, cumbersome steps, etc., to prevent changes in the three-dimensional structure of proteins. , Simple preparation method, simple application effect

Active Publication Date: 2019-11-12
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the difficulty of modifying traditional solid-phase materials to stabilize immobilized proteins, the steps are cumbersome, it is not conducive to production, the cost is greatly increased, and the limitations of cross-platform applications are not available. The traditional method of stabilizing solid-phase surface proteins has cumbersome steps. , very time consuming, very limited improvement in stability

Method used

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  • High-molecular polymer for stabilizing immobilized protein, and preparation method and application thereof
  • High-molecular polymer for stabilizing immobilized protein, and preparation method and application thereof
  • High-molecular polymer for stabilizing immobilized protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of novel polymer polyethylene glycol-alkyl alcohol-bovine serum albumin-dextran (mPEG-BSA-Hexadecanol-Dextran, PBHD)

[0057] 1. Preparation of aminated dextran

[0058] The preparation flow chart is as figure 1 shown.

[0059] 1-1) Dissolve 1 g of Dextran T70 in 10 ml of acetic acid buffer solution, stir magnetically to fully dissolve, add 0.8 g of sodium periodate, protect from light, and react at room temperature for 30 min. Then add 1.1g of sodium sulfite to terminate sodium periodate, react at room temperature for 10min, then use a 10KD ultrafiltration tube for ultrafiltration, change the liquid three times with a 15mMol / L carbonic acid buffer solution with pH=9, and redissolve to a volume of 10ml for subsequent use use.

[0060] 1-2) Add 5g of lysine to 10ml of formylated dextran, stir evenly, and react at room temperature for 2 hours. Add sodium hydride to the dextran derivative, react at room temperature for 30 minutes in the dark, and ...

Embodiment 2

[0082] Embodiment 2 Preparation of a complex solution of stable immobilized protein

[0083] The complex solution of the present invention is configured as follows: Weigh 375 mg of glycine-hydrochloric acid and dissolve it in 80 ml of water, then add 0.8 g of sodium chloride, add 0.1 ml of KroVin 300, and take 5 ml of 10% polyethylene glycol-alkane prepared in Example 1 of the present invention. Add alcohol-bovine serum albumin-dextran complex (PBHD) into the solution, stir well, mix evenly, adjust the pH to 7.4 with 4N NaOH, set the volume to 100ml, and store at 4°C.

Embodiment 3

[0084] Example 3 Verification of the stability of solid-phased antibodies

[0085] Take MYO R2 latex reagent, which is reagent A, take 8ml, centrifuge at 20000g for 30min, reconstitute with 4ml of the reconstitution solution prepared in Example 2, reconstitute with 20% power ultrasonic, disperse evenly, and age at 42°C for 4h, which is For reagent B, take 8ml of MYO R2 latex reagent and directly add 0.4ml of 10% polyethylene glycol-alkyl alcohol-bovine serum albumin-dextran complex solution (prepared in Example 1), and mix evenly to obtain reagent C. Then take three R1 20ml tubes respectively, which are reagents a, b, and c, corresponding to reagents A, B, and C, and conduct an accelerated 30-day test at 37 degrees Celsius.

[0086] Use the AU680 biochemical analyzer for detection. After the reagent parameters are set, put reagents A, B, and C into the instrument for testing. The reaction process is as follows: mix the R1 reagent and the calibrator evenly, incubate at 37°C fo...

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Abstract

The invention discloses a high-molecular polymer for stabilizing immobilized protein, and a preparation method and application thereof. The high-molecular polymer is a polyethylene glycol-alkyl alcohol-bovine serum albumin-glucan compound having a structure as shown in a formula I which is described in the specification. The preparation method comprises the steps of: 1) synthesis of aminated glucan; 2) preparing polyethylene glycol-p-nitrophenyl chloroformate and alkyl alcohol-p-nitrophenyl chloroformate compounds; 3) synthesis of a polyethylene glycol-alkyl alcohol-glucan compound; and 4) preparation of the polyethylene glycol-alkyl alcohol-bovine serum albumin-glucan compound. According to the invention, stability tests for low value and high value quality control through accelerated aging tests at 37 DEG C are conducted, and test results show that the individual high-molecular polymer provided by the invention or a reconstitution solution of the high-molecular polymer can greatly enhance the stability of reagents; and the preparation method is relatively simple, a process route is mature, application is easy, the preparation of the high-molecular polymer is convenient, and the high-molecular polymer is easy to store.

Description

technical field [0001] The invention relates to a high molecular polymer used for stabilizing immobilized proteins, and also relates to a preparation method of the high molecular polymer and an application in stabilizing solid phased proteins. The invention belongs to the technical field of biochemistry. Background technique [0002] Antigen-antibody specific binding is widely used in biochemical detection (latex immunoturbidity), immunoassay (magnetic particle luminescence, plate chemiluminescence, POCT), protein chip, biosensor and biochip, biomolecular engineering, material chemistry Advances in chemistry with cross-linking agents have greatly promoted the development of antibody immobilization technology, so the immobilization of antibodies has been developed very maturely, but the stability of immobilized proteins is an important link in the development of diagnostic reagents. Antibodies can be immobilized to different types of solid surfaces by physical adsorption, co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G81/00G01N33/543G01N33/68
CPCC08G81/00G01N33/68G01N33/54306G01N33/54313
Inventor 胡国庆
Owner BEIJING LEADMAN BIOCHEM
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