Preparation method of hepatitis B antibody fragment, hepatitis B antibody fragment, kit and application

An antibody fragment, hepatitis B technology, applied in the preparation of hepatitis B antibody fragments, hepatitis B antibody fragments, kits and application fields, can solve the problems of side reactions, non-specific binding false positive diagnosis and so on

Pending Publication Date: 2021-12-10
普十生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in medical practice, when the existing hepatitis B antibody is applied to detection, some side effects often occur, for example, non-specific binding leads to false positive diagnosis in clinical practice, etc.

Method used

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  • Preparation method of hepatitis B antibody fragment, hepatitis B antibody fragment, kit and application
  • Preparation method of hepatitis B antibody fragment, hepatitis B antibody fragment, kit and application
  • Preparation method of hepatitis B antibody fragment, hepatitis B antibody fragment, kit and application

Examples

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preparation example Construction

[0034] The preparation method of the hepatitis B antibody fragment provided by the application comprises the following steps:

[0035] Add pepsin, dithioerythyl sugar alcohol and 2-mercaptoethanol to the hepatitis B antibody solution, and incubate for the first time; then add glycine, and perform purification after the second incubation. By using pepsin to digest hepatitis B antibody to improve the affinity of the antibody and reduce non-specific binding.

Embodiment 1

[0036] Example 1 Preparation of Hepatitis B Antibody Fragments

[0037] In this embodiment, the following process parameters are taken as examples for illustration.

[0038] 1. Dilute pepsin (Beijing Suolaibao, product number: P8160, 10000U / mg) with citric acid buffer (0.06mol / L pH4.8) to 1mg / ml, adjust the pH value to 2.0 with concentrated hydrochloric acid, and set aside.

[0039] 2. Take 2ml of hepatitis B antibody (Fei Peng Bio, batch number: 20200518, 5mg / ml) into a 50KD dialysis bag, and dialyze with 100ml of citric acid buffer (0.06mol / L pH4.8) for 30min. After dialysis, cut 1ug of hepatitis B antibody with 1U pepsin, add 0.5ml of the above pepsin dropwise, then add 0.005ml of 5mM dithioerythyl sugar alcohol DTE (1.5g / L) and 0.1ml of 100mM 2-mercaptoethanol (0.3g / L), put the mixture in an oven at 25°C, incubate for 20min, then add 75ul glycine (3.3g / L) with a concentration of 1.5mol / L, and rotate at room temperature for 10min to react. After the reaction, the pH value...

Embodiment 2

[0043] Example 2 Detection of hepatitis B antigen

[0044] The hepatitis B antigen was detected by using the hepatitis B antibody fragment (fragment for short) in Example 1. There are many methods for detecting antigens by using antibodies, and colloidal gold testing is used as an example for illustration in this embodiment.

[0045] Membrane coating solution: Tris 1.21g, sucrose 0.5g, EDTA-2Na 0.5g, SDS 0.15g, hydrochloric acid 200ul, dilute to 100ml water.

[0046] Colloidal gold complex solution: Tris 1.21g, sucrose 2.5g, polyvinylpyrrolidone (PVA) 0.05g, BSA 0.5g, polyethylene glycol 0.05g, sodium caseinate 0.5g, hydrochloric acid 200ul, Tween 20 0.5ml , Dilute to 100ml of water.

[0047] Conjugate pad treatment solution: KH 2 PO 4 0.02g, TW20 0.1ml, Na 2 HPO 4 0.29g, 10g sucrose, 1g BSA, 0.05ml PC, dilute to 100ml water.

[0048] Sample pad treatment solution: KH 2 PO 4 0.02g, Na 2 HPO 4 12H 2 O 0.29g, KCl 0.02g, PC 0.05ml, TW201ml, Trition100 0.1ml, dilute t...

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Abstract

The invention belongs to the technical field of medical instruments, and particularly discloses a preparation method of a hepatitis B antibody fragment, the hepatitis B antibody fragment, a kit and an application, wherein the preparation method comprises the following steps: adding pepsin, dithioerythritol and 2-mercaptoethanol into a hepatitis B antibody solution, and incubating for the first time; and adding glycine, and purifying after secondary incubation. The preparation method at least has one of the following beneficial effects: when the hepatitis B antibody fragment prepared by the preparation method of the hepatitis B antibody fragment provided by the invention is applied to detection of hepatitis B antigen, the generation of false positive can be remarkably reduced, so that the accuracy of a detection result is improved.

Description

technical field [0001] The application belongs to the technical field of antibodies and their preparation, more specifically, it relates to a method for preparing hepatitis B antibody fragments, hepatitis B antibody fragments, kits and applications. Background technique [0002] Hepatitis B surface antigen (HBsAg) is the coat protein of hepatitis B virus. It is not infectious in itself, but its appearance is often accompanied by the presence of hepatitis B virus, so it is a sign of hepatitis B virus infection. It can exist in patients' blood, saliva, breast milk, sweat, tears, nasopharyngeal secretions, semen and vaginal secretions. Positive results can be detected in serum 2 to 6 months after infection with hepatitis B virus, and 2 to 8 weeks before the elevation of alanine aminotransferase. Most patients with acute hepatitis B can turn negative early in the course of the disease, and this indicator can continue to be positive in patients with chronic hepatitis B. Therefo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/576G01N33/535
CPCG01N33/5764G01N33/535
Inventor 高希贤王琳
Owner 普十生物科技(北京)有限公司
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