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A method for discovering specific functional antibodies

A specific, antibody technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of difficult bias, difficult samples, inaccurate antibody gene spectrum information, etc. Low, high throughput, simplified build process effects

Active Publication Date: 2020-04-03
GENEWIZ INC SZ
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The characteristics of next-generation sequencing, such as read length, accuracy, throughput, and cost-effectiveness, are suitable for studying the diversity of antibody library composition. However, there are also some technical difficulties in antibody sequencing library construction, high-throughput antibody sequencing, and data analysis. The obtained antibody gene spectrum information is inaccurate, the contained antibody gene information is biased, it is difficult to accurately define the change of CDR3 frequency, and the efficiency of in vitro pairing function verification of the selected full-length genes is low
[0006] Next-generation sequencing has been widely used to study the diversity of antibody gene repertoire composition, but how to avoid introducing bias in the process of sequencing library construction has always been a difficult problem
However, it is difficult to obtain enough samples in actual work, especially the number of clinical and pathological samples is often difficult to meet the needs of RACE library construction

Method used

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  • A method for discovering specific functional antibodies
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  • A method for discovering specific functional antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 An improved high-throughput library construction method suitable for micro-sample

[0085] The specific steps of library construction are as follows: figure 2 As shown, sample preparation and first-strand cDNA synthesis: Peripheral blood mononuclear cells (PBMC) isolated from human or animal peripheral blood by density gradient centrifugation. Total RNA was isolated from PBMC using Trizol. Take 20ng, 100ng and 600ng of RNA as the template and oligo(dT) as the primer for the synthesis of the first-strand cDNA. For specific operations, refer to the kit RACE 5' / 3'Kit instructions.

[0086] The optimized two-step PCR method constructs the antibody library, the first round of PCR amplification, and the forward primer is optimized RACE 5' / 3'Kit UPM, containing part of the Illumina adapter primer, the reverse primer contains the sequence of the antibody constant region and part of the Illumina adapter primer. VH, VK and VL are amplified separately, and the rea...

Embodiment 2

[0098] Example 2 Using an optimized antibody gene screening method to discover monoclonal antibodies against dengue fever

[0099] Using the RACE antibody library construction method described in Example 1, total RNA was isolated from PBMCs in the acute phase and recovery phase of dengue patients, and heavy chain, Kappa chain, and Lambda chain RACE libraries were respectively constructed. The library was sequenced using the Illumina Miseq 2×300 system, the heavy chain library was sequenced separately, and the Kappa and Lambda chain libraries were combined and sequenced. The sequencing results were processed with the software Trimmomatic-0.30, and the specific parameters were set as follows: phred33, LEADING: 20, TRAILING: 20, SLIDINGWINDOW: 20: 20, MINLEN: 200) to remove data with poor quality. The following improvements were made to the data analysis. Comparing the data volume of the antibody library with the singleton sequence removed and retained, it was found that the dat...

Embodiment 3

[0130] Example 3 Discovering Influenza Monoclonal Antibodies Using the Optimized Antibody Gene Screening Method

[0131] Using the RACE antibody library construction method described in Example 1, total RNA was isolated from PBMCs of volunteers before and 7 days after influenza vaccine injection, and the heavy chain, Kappa chain and Lambda chain RACE libraries were respectively constructed according to the method described in Example 2 ;Using Illumina Miseq 2×250 system for high-throughput sequencing. The processing of data, the method of determining FR and CDR regions, and the method of selecting candidate CDR3 amino acid sequences are all the same as those described in Example 1.

[0132] Table 10 Frequency Analysis of Heavy Chain CDR3 Before and After Influenza Vaccine Immunization

[0133]

[0134]

[0135] Table 11 Frequency analysis of Kappa chain CDR3 before and after influenza vaccine immunization

[0136]

[0137] Table 12 Frequency Analysis of Lambda Chain...

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Abstract

The invention relates to a method for discovering specific functional antibodies, in particular to a method for discovering specific functional antibodies based on sequence composition and frequency analysis of the variable region of an immune host antibody. Compared with conventional high-throughput antibody sequencing methods, this method can quickly and accurately obtain the full-length sequence of antibody variable regions including candidate CDR3, has a high success rate of antibody gene pairing and is suitable for detecting trace samples, and improves the ability to obtain specific functional antibodies. s efficiency.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for discovering specific functional antibodies, in particular to a method for discovering specific functional antibodies based on the composition and frequency analysis of the variable region of immune host antibodies. Background technique [0002] Antibodies produced by a single B cell that are completely identical and only target a specific epitope are called monoclonal antibodies. Since Kohler and Milstein reported using hybridoma technology to obtain monoclonal antibodies against sheep red blood cells in 1975, more and more monoclonal antibodies have been widely used in the fields of laboratory medicine diagnosis and separation of biological macromolecules. With the advantages of strong specificity, high purity, and good uniformity, monoclonal antibodies have improved the efficiency and accuracy of medical testing, and at the same time, they have also greatly pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/13C07K16/28
CPCC07K16/2803C12N15/1096C07K16/1018C07K16/1081C07K16/005C07K2317/10C07K2317/565C40B50/06C40B40/08C07K16/18C12N15/10C40B30/04G16C20/60G16B15/00G16B35/00C07K2317/21C07K2317/567C12N15/1062C12N15/1089
Inventor 陈维之吴昕王望孙中平李世红
Owner GENEWIZ INC SZ
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