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Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment

A technology of monoclonal antibody and gonadotropin, which is applied in the direction of fixing on/in organic carrier, fermentation, etc., can solve the problems of removing free enzymes to reduce product recovery rate, free enzymes cannot be reused, etc., and achieve fewer purification steps , Simple recovery and high purity

Inactive Publication Date: 2012-04-18
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] According to current literature and patent reports at home and abroad, antibody fragments are prepared by hydrolyzing antibodies with free papain or pepsin and then undergoing multi-step separation and purification. This type of method has free enzymes that cannot be reused, and the removal of free enzymes will also reduce Product recovery and other issues

Method used

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  • Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment
  • Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment
  • Method for preparing anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment

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Embodiment 1

[0030]1) Immobilization of papain: cut the filter paper into pieces, add tap water, boil, boil, filter with 8 layers of gauze, take the filter cake and add 1000ml 0.1mol / L NaOH solution, stir for 2 hours, wash with water until the pH is close to 7.0, pump Filter, get 1.2g filter cake (dry weight) and add 1mol / L NaOH solution in the ratio of 1:9 (g / ml), then add Epichlorohydrin, stirred and reacted in a water bath at 60°C for 30 minutes, suction filtered, the filter cake was fully washed with deionized water, and drained, and p-phenylenediamine (0.24 g p-phenylenediamine phenylenediamine / 1.2g filter cake carrier), reacted at 45°C for 30min, filtered, the filter cake was fully washed with distilled water, and vacuum-dried to constant weight to obtain 1.2g of ammoniated paper fiber. Prepare immobilized papain with ammoniated paper fiber as a carrier and glutaraldehyde as a crosslinking agent: disperse the above 1.2g ammoniated paper fiber in 0.1mol / L acetate buffer (containing 1m...

Embodiment 2

[0035] 1) Immobilization of papain: Cut the filter paper into pieces, add tap water, boil, filter with 8 layers of gauze after boiling, add 1000ml of 0.2mol / L NaOH solution to the paper fiber, stir for 3 hours, wash with water until the pH is close to 7.0, Suction filtration, get 1.2g filter cake (dry weight) add the NaOH solution of 1mol / L by the ratio of 1:9 (g / ml), then make up by the ratio of 1:1 (g / ml) with filter cake mass volume ratio. Add epichlorohydrin, stir and react in a water bath at 60°C for 30 minutes, filter with suction, wash the filter cake fully with deionized water and then drain it to obtain a filter cake, then add p-phenylenediamine at a mass volume ratio of 0.2:1 to 1.2 g of the filter cake (0.24g p-phenylenediamine / 1.2g filter cake carrier), react at 45°C for 30min, fully wash with distilled water, and vacuum dry to constant weight to obtain ammoniated paper fiber.

[0036] Prepare immobilized papain with ammoniated paper fiber as carrier, glutaraldehyd...

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Abstract

The invention discloses a method for preparing an anti-human chorionic gonadotrophin (hCG) monoclonal antibody fragment. The method comprises the following steps of: putting activated immobilized papain into a 0.1mol / L acetic acid buffer solution with pH of 5.0, adding an anti-hCG monoclonal antibody, performing water bath reaction for 4 to 24 hours at the temperature of between 30 and 60 DEG C, and filtering the reaction solution, wherein filter cakes B are immobilized papain and recycled, and filtrate B is a hydrolysate containing the monoclonal antibody fragment; and separating and purifying the hydrolysate containing the monoclonal antibody fragment by adopting weak anion exchange chromatography, and thus obtaining the anti-hCG monoclonal antibody fragment. The method is easy to implement and has a few purification steps, and the obtained antibody fragment has high purity; and the antibody fragment prepared in the method can reach the purity of over 90 percent by one-step 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 an anti-human chorionic gonadotropin monoclonal antibody fragment. (2) Background technology [0002] Human chorionic gonadotrophin (hCG) is a glycoprotein hormone secreted by syncytiotrophoblast cells of human placenta after conception, with a molecular weight of about 38000D. The synthesis and secretion of hCG are closely related to pregnancy. The examination of hCG can be used as an important indicator of early pregnancy detection. Using anti-hCG monoclonal antibody for early pregnancy detection can further improve the sensitivity and specificity of early pregnancy diagnosis. Moreover, the content of hCG is closely related to many tumor diseases, and hCG can be regarded as one of the tumor markers. Therefore, the examination of hCG can also provide a basis for the diagnosis and treatment monitoring of trophoblastic tumors and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C12N11/12
Inventor 应国清李慧娟王鸿易喻梅建凤陈建澍
Owner ZHEJIANG UNIV OF TECH
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