Method for preparing AFP (alpha fetoprotein) monoclonal antibody by in-vitro culture and application of AFP monoclonal antibody

A monoclonal antibody and in vitro culture technology, applied in instruments, analytical materials, measuring devices, etc., can solve the problems of easy pollution, troublesome manual operation, etc., and achieve the effects of long-term growth, simplified operation, and optimized process

Inactive Publication Date: 2019-06-18
ZHENGZHOU IMMUNO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a new method for in vitro culture and preparation of AFP monoclonal antibody. The method adopts an improved hollow fiber culture system for culture and preparation of antibodies, which solves the problems of troublesome manual operation and easy pollution, and can meet the detection requirements of chemiluminescent methods. Required monoclonal antibody requirements

Method used

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  • Method for preparing AFP (alpha fetoprotein) monoclonal antibody by in-vitro culture and application of AFP monoclonal antibody
  • Method for preparing AFP (alpha fetoprotein) monoclonal antibody by in-vitro culture and application of AFP monoclonal antibody
  • Method for preparing AFP (alpha fetoprotein) monoclonal antibody by in-vitro culture and application of AFP monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of AFP monoclonal antibody by in vitro culture using hollow fiber system

[0025] The first step is to recover hybridoma cells and prepare seed cells

[0026] (1) Before the experiment, the ultra-clean workbench needs to be disinfected for 30 minutes, the wind speed is set to level 3, ventilated for 15 minutes, the measured wind speed is greater than 0.3m / s, and the equipment usage records and wind speed measurement records are filled in. Wipe and disinfect the surface of the ultra-clean workbench, required consumables, the surface of reagent bottles and hands with 75% alcohol.

[0027] (2) Preheat the required DMEM medium and serum in a 37°C water bath, and then configure DMEM medium containing 15% newborn bovine serum (collectively referred to as complete medium) in the ultra-clean workbench.

[0028] (3) After the hybridoma cells were taken out from the liquid nitrogen, the cell suspension was sucked out, and slowly added to a centrifuge tube p...

Embodiment 2

[0048] Example 2 Purification of AFP monoclonal antibody (purify the in vitro antibody sample to be purified harvested in Example 1)

[0049] 1. Harvested in vitro antibody sample processing

[0050] (1) First detect the concentration of the in vitro antibody sample to be purified: take 2 microliters of the sample and use the UV spectrophotometer IgG mode to detect its content, and the blank control is 0.02M PBS with pH 7.4.

[0051] (2) Pass the collected in vitro antibody samples through a 0.45 μm filter membrane.

[0052] 2. Purification process

[0053] (1) Initial pump washing pipeline: Open the PrimeView software, put all the inlets of AKTAprime plus into filtered and purified water, operate the machine, enter Templates / Application Template / System Wash Method in turn, select B, A1, A3, A4, A8, press the "OK" button, the system will automatically flush.

[0054] (2) Place the injection ports of all pipelines into corresponding reagent bottles;

[0055] (3) Pump and wa...

Embodiment 3

[0061] Example 3 Performance evaluation of the AFP monoclonal antibody prepared by the present invention

[0062] The chemiluminescent AFP detection kit is used for detection. The principle of the kit is the double-antibody sandwich method to detect antigens. The monoclonal antibodies (hereinafter referred to as in vitro antibodies) prepared by the present invention are used as coating antibodies and labeled antibodies respectively, and compared with the kits in production. .

[0063] 1. Sensitivity assessment

[0064] Measure the zero point (S0) of 10 standard products, calculate the average value of luminescence and its standard deviation, and then bring the value obtained by adding twice the standard deviation (SD) of the average value of luminescence (`X) into the standard curve to obtain the corresponding concentration value. The sensitivity test is shown in Table 1 below.

[0065] Table 1 Sensitivity comparison of in-produced antibody and in vitro antibody preparation...

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Abstract

The invention discloses a method for preparing an AFP (alpha fetoprotein) monoclonal antibody by in-vitro culture. The method comprises steps as follows: firstly, hybridoma cells are resuscitated, andseed cells are prepared; a liquid inlet end of a sterile hollow fiber system internal circulatory system is connected with a PBS (polybutylene succinate) / medium supply bottle, a liquid outlet end isconnected with a liquid waste bottle, a liquid inlet end of a hollow fiber system external circulatory system is connected with the PBS / medium supply bottle, and a liquid outlet end is connected withthe liquid waste bottle, and a hollow fiber system is rinsed by PBS and a medium; then the seed cells are imported; glucose and lactic acid are monitored continuously in a culturing process, metabolicwaste is removed, meanwhile, a fresh medium is supplemented continuously, a 20L bucket is adopted for storing the medium, and operation frequency is reduced; after an obtained cultural supernatant issubjected to affinity purification, a finished monoclonal antibody product with antibody concentration being 5 mg / mL and purity being 95% or higher is obtained. The prepared monoclonal antibody is good in specificity, high in sensitivity and high in potency, and sensitivity, specificity and stability of a kit can be greatly improved when the monoclonal antibody is applied to an AFP chemiluminescence detection kit.

Description

technical field [0001] The present invention relates to in vitro diagnostic reagents, in particular to a method for in vitro culture and preparation of AFP monoclonal antibodies, and also relates to the application of the prepared monoclonal antibodies in tumor marker ELSA detection diagnostic kits. Background technique [0002] Since the discovery of alpha-fetoprotein (AFP) in the 1960s, AFP has been used as an important indicator for the screening and diagnosis of primary liver cancer (HCC). After nearly half a century of research, researchers have gradually deepened their understanding of the physical and chemical properties, biological functions and clinical applications of AFP. Alpha-fetoprotein (AFP) is a glycoprotein that belongs to the albumin family and is mainly synthesized by fetal liver cells and yolk sac. Alpha-fetoprotein has a high concentration in fetal blood circulation, and it decreases after birth. Alpha-fetoprotein is basically replaced by albumin in 2-3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/08G01N33/577G01N33/574
Inventor 孙静静程雷显周伟伟周雷鸣赵巧辉李桂林付光宇吴学炜
Owner ZHENGZHOU IMMUNO BIOTECH
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