Method for separating human chorionic gonadotropin monoclonal antibody segment

A technology of chorionic gonadotropin and monoclonal antibody, which is applied in the direction of immobilization on/in it, fermentation, etc., and can solve the problem of reduced catalytic efficiency

Inactive Publication Date: 2015-01-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional immobilized carriers all carry out heterogeneous catalysis, which makes the enzyme have certain steric hindrance in the catalytic process, resulting in a decrease in the catalytic efficiency of the enzyme. However, the immobilized enzyme on the intelligent polymer carrier can achieve homogeneous phase by controlling the reaction conditions. Catalysis and heterogeneous separation, improving the catalytic efficiency and reuse rate of the enzyme

Method used

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  • Method for separating human chorionic gonadotropin monoclonal antibody segment
  • Method for separating human chorionic gonadotropin monoclonal antibody segment
  • Method for separating human chorionic gonadotropin monoclonal antibody segment

Examples

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Embodiment 1

[0029] 1) Immobilization of papain: Weigh 10 mg of NHS-PNIPAM and dissolve it in 100 μL of N,N-dimethylformamide that has been dried overnight with activated 4A molecular sieves (that is, 4A molecular sieves are activated at 300 ° C for 4 hours, and then added after cooling In N,N-dimethylformamide, separate layers overnight, take the upper layer), add 500μL, 2mg / ml papain solution (prepared with boric acid buffer solution with pH 8.5, 0.1mol / L, the specific enzyme activity of papain 570.3U / mg), shake in a water bath at 10°C for 2h, dilute the resulting reaction solution with pH 7.0, 0.05mol / L PBS buffer solution to 4 times the volume, centrifuge at 11000r / min at 40°C for 30min, collect the precipitate, and use a 4°C pH 7.0, dissolved in 0.05mol / L phosphate buffer, after the precipitate was completely dissolved, centrifuged at 40°C, 11000r / min for 30min, this process was repeated 3 times (i.e. dissolved, centrifuged 3 times), and the precipitate was collected for immobilization...

Embodiment 2

[0042] 1) Immobilization of papain: Weigh 10 mg of NHS-PNIPAM and dissolve it in 100 μL of N,N-dimethylformamide dried overnight with activated 4A molecular sieve (the method is the same as in Example 1), add 500 μL, 20 mg / ml Papain solution (prepared with pH 8.5, 0.1mol / L boric acid buffer solution, 570.3U / mg), shake in a water bath at 25°C for 10h, and dilute the resulting reaction solution to 4 times with pH 7.0, 0.05mol / L PBS buffer solution Volume, centrifuge at 11000r / min at 40°C for 30min, collect the precipitate, dissolve it with pH 7.0, 0.05mol / L phosphate buffer at 4°C, after the precipitate is completely dissolved, centrifuge at 11000r / min at 40°C for 30min, repeat the process 3 times (dissolving and centrifugation repeated 3 times), the collected precipitate is immobilized papain (78.5U / mg carrier, specific enzyme activity 245.3U / mg), and finally dissolved in the newly prepared pH 7.0, 0.05mol / L Store in phosphate buffer at 4°C to obtain immobilized papain solution...

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Abstract

The invention discloses a method for separating a human chorionic gonadotropin monoclonal antibody segment. The method comprises the following steps: dissolving a temperature sensitive carrier NHS-PNIPAM immobilized papain into a 0.05mol / L PBS buffer solution with the pH value of 7.0, adding a human chorionic gonadotropin monoclonal antibody, reacting for 2-24 hours at a water bath of 20-60 DEG C, centrifuging a reaction liquid for 15 minutes at the speed of 10000r / min at 40 DEG C, recycling precipitate, namely NHS-PNIPAM immobilized papain, collecting filtrate, namely hydrolysate containing an antibody Fab segment, and performing separation purification on the hydrolysate containing the antibody segment by using protein L affinity chromatography, thereby obtaining the antibody Fab segment. The method is simple and convenient to operate, small in purification step and high in antibody segment purity; the antibody Fab segment prepared by using the method can achieve the purity greater than 96.0% by virtue of only one step of purification.

Description

(1) Technical field [0001] The invention relates to a preparation method of a monoclonal antibody fragment, in particular to a preparation method of a human chorionic gonadotropin monoclonal antibody fragment. (2) Background technology [0002] Human chorionic gonadotrophin (HCG) is a glycoprotein hormone secreted by humans after conception to maintain normal pregnancy. Pregnancy can be confirmed by detecting HCG in urine. In addition, studies have found that the synthesis and secretion of HCG are also related to gynecological diseases and malignant tumors. Therefore, HCG monoclonal antibodies can not only be used for early pregnancy diagnosis, but also have a certain role in the diagnosis and identification of pregnancy-related diseases and malignant tumors. reference value. However, the complete HCG monoclonal antibody has a large molecular weight and complex structure, poor penetration in tissues, and the presence of the Fc fragment is likely to cause heterogeneous immun...

Claims

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Application Information

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IPC IPC(8): C12P21/06C12N11/08
Inventor 易喻应国清高虹王鸿梅建凤陈建澍
Owner ZHEJIANG UNIV OF TECH
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