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A screening method for trace antibodies in the supernatant stage of hybridoma cells

A hybridoma cell and screening method technology, which is applied in the field of screening trace antibodies in the supernatant stage of hybridoma cells, can solve the problems of serum effect difference, blind preparation, etc., achieve shortened preparation time, high cost, and avoid expanded culture and protein production. The effect of the purification work

Active Publication Date: 2015-09-09
无锡国盛生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the prepared monoclonal antibody may not be able to perform double-antibody sandwich pairing against the immunogen, and there is a certain degree of blindness in the preparation.
Even if antibodies that can be paired with double antibodies against the immunogen are screened out, there are still large differences in clinical serum effects

Method used

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  • A screening method for trace antibodies in the supernatant stage of hybridoma cells
  • A screening method for trace antibodies in the supernatant stage of hybridoma cells
  • A screening method for trace antibodies in the supernatant stage of hybridoma cells

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Screening of Trace Antibodies in the Supernatant Stage of Hybridoma Cells

[0033] 1. Take the culture supernatant of the hybridoma cells formed after cell fusion, remove impurities by salting out and replace the buffer system to obtain the pretreatment supernatant. The specific steps are as follows:

[0034] a) Add equal volumes of saturated ammonium sulfate solution to 29 cell supernatants, precipitate at 4°C for 15 hours, centrifuge at 12000 g for 10 min, and discard the supernatant to obtain the precipitate;

[0035] b) The precipitates were dissolved in phosphate buffer with a concentration of 0.01mol / L and a pH of 7.2, respectively, and placed in millipore ultrafiltration tubes with a volume of 0.5ml and a molecular weight of 50KD, centrifuged at 12000g for 10min, and repeated ultrafiltration for 2 times to obtain the pretreatment supernatant;

[0036] 2. Immobilize the supernatant antibody pretreated in step 1 to the microlens array chip:

[0037] a)...

Embodiment 2

[0052] 1. Take the culture supernatant of hybridoma cells formed after cell fusion, remove impurities by salting out and replace the buffer system to obtain the pretreatment supernatant:

[0053] a) 20 parts of cell supernatant were added to 150% saturated ammonium sulfate solution of the supernatant volume respectively, precipitated at 4°C for 8 hours, centrifuged at 12000g for 10min, discarded the supernatant to obtain the precipitate;

[0054] b) The precipitates were dissolved in phosphate buffer with a concentration of 0.01mol / L and a pH of 7.2, respectively, and placed in millipore ultrafiltration tubes with a volume of 0.5ml and a molecular weight of 50KD, centrifuged at 12000g for 10min, and repeated ultrafiltration for 2 Obtain pretreatment supernatant after times;

[0055] 2. Fix the supernatant protein pretreated in step 1 to the microlens array chip, the specific steps are as follows:

[0056] a) The pretreatment supernatant in step 1 is diluted 5 times with a spo...

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Abstract

The invention provides a method for screening micro antibodies in a hybridoma cell supernatant stage. The screening method comprises the following steps: pretreating supernatant, performing sample application and after-treatment on the supernatant, labeling the supernatant, testing sample application and performing double antibody sandwich detection. The micro antibodies are paired and screened in the hybridoma cell supernatant stage, the high-sensitivity and multi-flux advantages of a chip are utilized, and a plurality of objectless monoclonal antibody purification operations are avoided. When the type of monoclonal antibodies are prepared, paired antibodies can be screened even the paired antibodies with good clinical detection effects can be prescreened by using the method, the large-scale preparation is performed, the efficiency is greatly improved, and the preparation cost and risk are reduced.

Description

technical field [0001] The invention relates to an antibody screening method, in particular to a screening method for trace antibodies in the supernatant stage of hybridoma cells. Background technique [0002] The double-antibody sandwich method is the most commonly used ELISA method for clinical in vitro diagnostic detection of antigens, and is suitable for the quantitative detection of multivalent antigens with at least two antigenic determinants in the detection molecule. Its basic working principle is: use the antibody and enzyme-labeled antibody connected to the solid-phase carrier to combine with the two epitopes on the detected antigen molecule in the sample respectively to form a solid-phase antibody-antigen-enzyme-labeled antibody immune complex. [0003] The traditional classic double-antibody sandwich target antibody screening method needs to use hybridoma technology to prepare monoclonal antibodies (abbreviated as monoclonal antibodies). For relative affinity de...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/68
CPCG01N33/531G01N33/6803
Inventor 王振宇陆冬雷戴良
Owner 无锡国盛生物工程股份有限公司
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