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Method for opening the V domain of natural β2 glycoprotein I and antigen prepared by using the method, application and kit

A glycoprotein and structural domain technology, applied in the field of antigen preparation, can solve the problems of low sensitivity, high cost, and poor stability, and achieve the effect of simple and efficient method

Active Publication Date: 2022-02-08
ZHUHAI LIVZON DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the naturally extracted unprocessed β2 glycoprotein I antigen has a closed ring structure, the key V domain is not opened, the sensitivity is low, and it is easy to cause missed detection
β2 glycoprotein I has multiple disulfide bonds to form a complex spatial conformation, and the recombinant β2 glycoprotein I antigen often cannot reproduce such a complex conformation, so the reactivity is poor, the stability is not as good as that of the natural β2 glycoprotein I antigen, and the cost is also low. Much higher than the native β2 glycoprotein I antigen

Method used

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  • Method for opening the V domain of natural β2 glycoprotein I and antigen prepared by using the method, application and kit
  • Method for opening the V domain of natural β2 glycoprotein I and antigen prepared by using the method, application and kit
  • Method for opening the V domain of natural β2 glycoprotein I and antigen prepared by using the method, application and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] A method for natural β2-glycoprotein I, V domain is opened, comprising the steps of:

[0067] Step 1. Select a molecular weight of 50kDa human type natural β2-glycoprotein I.

[0068] Step 2. The native β2 glycoprotein I containing 1.15M NaCl, pH is in 20mM HEPES 11.5 (hydroxyethylpiperazine B sulfuric acid) buffer, dialyzed at 4 ℃ 48 hours, the medium was changed at 24 hours.

[0069] Obtained in Step 3. Step 2 β2 glycoprotein I containing 0.15M NaCl, pH 7.4 to 20mM HEPES buffer, and dialyzed at 4 ℃ 8 hours.

[0070] Obtained in step 4. Step β2 glycoprotein I 3 was added bacterial lipid A, disaccharide I obtained protein mass β2 and bacterial lipid A ratio of 500: 1, vortexed vessel was placed at 37 ℃, the reaction for 1 hour at 1000rpm.

[0071] Obtained in step 5. Step 4 I do β2 glycoprotein isolated and purified by HiTrap Chelating column.

[0072] Step 6. Step 5 obtained after purification β2 glycoprotein I quantified by UV spectrophotometer, containing 0.15M NaCl, pH 7....

Embodiment 2

[0103] The present embodiment provides a method for opening a natural β2-glycoprotein I, V domains, different from Example 1 in that, in step 2, HEPES buffer concentration of 10mM.

Embodiment 3

[0105] The present embodiment provides a method for opening a natural β2-glycoprotein I, V domains, different from Example 1 in that, in step 2, HEPES buffer concentration of 40mM.

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Abstract

The invention provides a method for opening the V domain of natural β2 glycoprotein I, an antigen prepared by the method, an application and a kit, and relates to the technical field of antigen preparation. The method for opening the V structural domain of natural β2 glycoprotein I of the present invention comprises the following steps: the natural β2 glycoprotein I is dialyzed for the first time, then mixed with bacterial lipid A, and reacted to obtain the β2 sugar opened by the V structural domain Protein I; the dialysate used in the first dialysis includes buffer A; the buffer A contains sodium ions at a concentration of 0.75-2M, and the pH is 9-12. Compared with the natural β2 glycoprotein I antigen and the recombinant β2 glycoprotein I antigen, the sensitivity of the β2 glycoprotein I with the V domain opened prepared by the method is greatly improved.

Description

Technical field [0001] Technical Field The present invention relates to method of preparing antigen, more particularly to a natural open β2 glycoprotein I and the domain V of antigen use, the application and the method of preparation of a kit. Background technique [0002] Anti-β2-glycoprotein I antibodies is an important laboratory parameters antiphospholipid syndrome classification standard, now widely used in clinical, clinical laboratory has become one of the most common autoantibodies project. And β2-glycoprotein I is detected β2 glycoprotein I Antibody Kit core key raw materials. [0003] Β2 glycoprotein I native molecular weight of about 50kD, of 326 amino acid residues and contains five domains, from amino terminus to carboxy terminus were DⅠ, DⅡ, DⅢ, DⅣ and DⅤ region. Before β2 glycoprotein I 4 highly homologous domains, each containing approximately 60 amino acid residues, districts have four cysteine ​​(Cys), consisting of two disulfide bonds. V domain of the β2 glycop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K1/34G01N33/68
CPCC07K14/4713G01N33/6893G01N2333/47G01N2800/24
Inventor 徐顺澍杨晶仪沈佳燕邓京
Owner ZHUHAI LIVZON DIAGNOSTICS
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