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Immunity enhancing method for Lp-PLA2, NGAL, and Derf24

A cationic carrier and protein technology, which is used in the field of Lp-PLA2, NGAL and Derf24 to enhance immunity, and can solve the problems that cannot meet the detection requirements

Active Publication Date: 2018-10-23
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, in the actual application process, we found that the antibody titers of Lp-PLA2, NGAL and Derf24 prepared by using c2BSA (that is, bovine serum albumin modified with ethylenediamine) as the carrier protein in the experiment of immunizing mice reached 1x10 respectively. 5 , 2x10 5 and 4x10 5 ; still can not meet the detection requirements

Method used

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  • Immunity enhancing method for Lp-PLA2, NGAL, and Derf24
  • Immunity enhancing method for Lp-PLA2, NGAL, and Derf24
  • Immunity enhancing method for Lp-PLA2, NGAL, and Derf24

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of cationized bovine serum albumin (c510BSA for short)

[0047] Use 1,5-pentanediamine and 1,10-decanediamine to prepare cationic bovine serum albumin by modifying natural bovine serum albumin. The specific steps of modification are as follows:

[0048] (1), add 200mg of 1,5-pentanediamine (CAS: 462-94-2) and 168.6mg of 1,10-decanediamine (CAS: 646-25-3) into 20ml H 2 In O, adjust the pH to 4.75 with 6N HCl, adjust the total volume to 50ml, and equilibrate to room temperature (25°C);

[0049] (2), add 737.2mg BSA (dissolved in 5ml H 2 in O);

[0050] (3), add 220 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter referred to as EDC, CAS: 25952-53-8) to the above solution, stir at room temperature for 1 Hour;

[0051] (4), prepare 4M pH=4.75 acetic acid buffer solution and add 3.5ml to terminate the reaction of the above solution;

[0052] (5), dialyze the above solution with 50mM pH=6MES;

[0053] (6) After measur...

Embodiment 2

[0054] Example 2: Coupling and synthesis of c510BSA-LP-PLA2 immunogen

[0055] The specific steps of coupling of cationized bovine serum albumin c510BSA and recombinant human lipoprotein-related phospholipase A2 are as follows:

[0056] (1), take 1ml of LP-PLA2 recombinant protein (R&D company product, 5mg) and dissolve it in 1ml MES (pH6.0) buffer solution, a total of three groups;

[0057] (2) Add 4.3 mg of EDC to the protein solution and react at room temperature for 5 minutes;

[0058] (3), with 0.5M Na 2 HPO 4 Adjust to pH=7.2;

[0059] (4), add 5.9 mg of N-hydroxysuccinimide (NHS) to the above solution and react at room temperature for 20 minutes;

[0060] (5), 2 mg of cationized bovine serum albumin c510BSA, nBSA (unmodified), and KLH were added to the protein solution and reacted for 1.5 hours;

[0061] (6) The protein coupling mixture was dialyzed into PBS (pH7.2), and the c510BSA-LP-PLA2 recombinant protein immunogen, nBSA-LP-PLA2 recombinant protein immunogen, ...

Embodiment 3

[0062] Example 3: The immune effect of the c510BSA-LP-PLA2 protein immunogen conjugate

[0063] (1), the prepared immunization antigen is the c510BSA-LP-PLA2 recombinant protein immunogen conjugate prepared in Example 2, the conjugate nBSA-LP-PLA2 of natural bovine serum albumin (nBSA) and LP-PLA2 recombinant protein , and the conjugate KLH-LP-PLA2 of hemocyanin (KLH) and LP-PLA2 recombinant protein;

[0064] (2), using the c510BSA-LP-PLA2, nBSA-LP-PLA2 and KLH-LP-PLA2 immunogens prepared in Example 2, adopt conventional methods to inoculate experimental animal mice (BALB / c) respectively, and take small mice after booster immunization. Mouse antiserum, the specific steps are as follows:

[0065] Dilute the c510BSA-LP-PLA2, nBSA-LP-PLA2 and KLH-LP-PLA2 immunogens synthesized above to 1 mg / ml with sterile water to obtain an antigen solution, and then use 1.0 ml of the antigen solution with an equal amount of Freund’s After the complete adjuvant was mixed, the experimental mice...

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Abstract

The invention belongs to the field of chemical modification of bovine serum albumin, and specifically relates to an immunity enhancing method for Lp-PLA2, NGAL, and Derf24. Bovine serum albumin, whichis modified by 1,5-pentanediamine and 1,10-decanediamine, is taken as the carrier protein and is coupled with recombinant lipoprotein associated phospholipase A2 (Lp-PLA2), recombinant neutrophil gelatinase associated lipocalin (NGAL), and recombinant dermatophagoides farinae allergen (Derf24) to prepare immunogen, which is used to immunize a mouse. An antibody titer with higher specificity titeris obtained, and the immunogen is used for diagnostic reagents.

Description

technical field [0001] The invention relates to the field of chemical modification of bovine serum albumin, in particular to a method for enhancing immunity of Lp-PLA2, NGAL and Derf24, which is mainly modified by 1,5-pentanediamine and 1,10-decanediamine As a carrier protein, bovine serum albumin was conjugated with recombinant human lipoprotein-associated phospholipase A2 protein (Lp-PLA2), recombinant neutrophil gelatinase-associated lipocalin (NGAL) and recombinant dust mite allergen Derf24 protein antigen, respectively. Combined to prepare immunogen for immunization of mice. Background technique [0002] Existing studies have shown that lipoprotein-associated phospholipase A2 (Lipoprotein-Associated PhospholipaseA2, Lp-PLA2) is a marker of vascular inflammation, and its activity and content are related to cardiovascular and cerebrovascular disease events caused by atherosclerotic plaques. There is a significant positive correlation. By detecting Lp-PLA2 in the blood, ...

Claims

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Application Information

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IPC IPC(8): C07K1/107C07K1/34C07K14/765C07K19/00
CPCC07K14/43531C07K14/765C12N9/18C12Y301/01004
Inventor 赵振富唐艳吉坤美陈家杰张真胡葭耘何永燊
Owner SHENZHEN UNIV
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