Efficient screening method of paired antibodies

A paired antibody and screening method technology, applied in the biological field, can solve the problems of low efficiency and high cost, and achieve the effect of simple steps, less loss and high affinity

Pending Publication Date: 2021-08-31
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, in terms of manpower, raw materials, and consumable costs, each positive monoclonal cell needs to go through steps such as clone amplification, mouse ascites preparation, ascites purification, labeling, and ELSIA pairing. The efficiency is low and the cost is relatively high.

Method used

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  • Efficient screening method of paired antibodies
  • Efficient screening method of paired antibodies
  • Efficient screening method of paired antibodies

Examples

Experimental program
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Embodiment 1

[0038] This embodiment provides a method for preparing a positive cloned cell line, which includes the following steps: using the new coronavirus S protein of the mammalian expression system as an antigen, emulsifying the antigen with Freund's adjuvant, injecting subcutaneously at multiple points, and immunizing Balb / c small 4 mice. After the first immunization, the complete Freund's adjuvant was mixed with the antigen 1:1 to form an oil packet, and each mouse was immunized with 50 μg of the antigen, and the immune volume was 1 mL; 3 weeks later, the emulsified antigen was boosted with incomplete Freund's adjuvant to boost the immunity. The immunization interval is 2 weeks, and a total of at least 4 immunizations have been passed. The quality of the antigen, the effect of emulsification, and the number of immunizations directly determine the titer of the antiserum of the animal after immunization; the higher the titer, the more positive clones will be in the end, and the numbe...

Embodiment 2

[0043] This example provides a method for efficiently screening high-affinity paired antibodies, including the following steps:

[0044] (1) 10 mL of the supernatants of the 22 positive clonal cells obtained in Example 1 were collected respectively.

[0045] (2) Use Pro G magnetic beads and Pro A magnetic beads respectively as mixed magnetic bead fillers (Protein A / G). Glycine solution was eluted, and then neutralized by adding 5 μL neutralizing solution pH 9.0 Tris-HCl solution, and the concentration of the antibody mixture obtained after purification and collection was shown in the table below:

[0046] Numbering clone number volume Concentration (mg / mL) 1 1B8D11 200μL 1.23 2 2F2D8 200μL 0.89 3 2G12F3 200μL 1.02 4 2C5D4 200μL 0.54 5 1B1C4 200μL 0.56 6 8G10G9 200μL 0.98 7 8G10B11 200μL 0.74 8 6F12C9 200μL 0.67 9 6B10E3 200μL 1.11 10 2H10B10 200μL 1.25 11 3B10D11 2...

Embodiment 3

[0075] This embodiment provides a screening method for paired antibodies of PDCoV-ORF1ab, comprising the following steps:

[0076] S1, the antigen PDCoV-ORF1ab protein was prepared by the Escherichia coli system, and the same method as in Example 1 was used for immunization, fusion, and preparation of positive clones, and a total of 14 positive clones were obtained. The 14 positive clonal cells that were frozen were collected for supernatant antibody titer detection, and the test results are shown in the table below:

[0077] Numbering clone number OD 450nm value

1 14G11G9 2.7826 2 14G11A11 2.6199 3 13F10A9 2.796 4 8E4B6 2.6384 5 9E3F7 2.4816 6 12F11F8 2.4203 7 12G6G9 2.4203 8 3H5E6 2.523 9 9C3C5 2.6867 10 11F12H6 2.122 11 6D12B5 2.6463 12 8E7F6 2.5436 13 3H9G4 2.5752 14 13E7E1 2.666 15 Positive serum 1:10000 2.1899 16 Negative SP2 / 0 supernatant 0.04...

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Abstract

The invention provides an efficient screening method of high-affinity paired antibodies, wherein the method comprises the steps: adsorbing antibodies in positive clone cell supernate through Protein A/G magnetic bead filler, measuring the concentration of the antibodies, selecting a preset amount of antibodies for labeling to form a detection antibody, coating unlabeled antibodies, performing an ELISA test by adopting an orthogonal chessboard method, and screening an experimental group with a high OD value and rechecking antibody pairs according to an ELISA result. According to the formula antibody screening, the whole experiment process from positive clone cell strain determination to antibody pairing is completed only in two days, and the average cost of consumable reagents is less than 100 Yuan, so that the optimal antibody pair can be efficiently obtained at low cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-efficiency screening method for paired antibodies. Background technique [0002] Paired antibodies (antibody pairs) refer to a pair of antibodies that can simultaneously bind to an antigen, and are mainly used in double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). [0003] The principle of antibody pairing is as follows: Usually, an antigen molecule has multiple antigenic determinants, and the antigen is injected into animals for immunization, and different antibodies will be produced against different antigenic determinants. The antibodies produced are specific and specific, that is, an antibody molecule can only specifically bind to one antigenic determinant. Two antibodies against different antigenic determinants may bind to the antigen molecule at the same time. Two antibodies are paired antibodies if they bind to the same antigen molecule at the same tim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6854
Inventor 张芳李晶晶王婷周文祥易汪雪
Owner CUSABIO TECH LLC
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