Antibody discovery method based on hybridoma technique and high-throughput sequencing

A hybridoma and high-throughput technology, applied in the field of biomedicine, can solve the problems of difficulty in obtaining high-affinity monoclonal antibodies, increase the possibility of obtaining functional antibodies, and the small range of antibody cell line screening, so as to overcome the blindness of pairing, Increased likelihood, highly reproducible effects

Inactive Publication Date: 2016-11-02
GENEWIZ INC SZ
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Problems solved by technology

[0006] In view of the shortcomings of the existing hybridoma technology, such as long time-consuming, limited screening range of antibody cell lines, and difficulty in obtaining high-affinity monoclonal antibodies, as well as defects such as library construction bias and pairing blindness in antibody sequencing technology, the present invention will optimize the high-pass The combination of quantitative antibody sequencing method and hybridoma antibody screening technology, compared with traditional hybridoma monoclonal antibody technology, expands the selection range of candidate antibody genes, improves the efficiency of antibody structure optimization, and increases the possibility of obtaining functional antibodies sex

Method used

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  • Antibody discovery method based on hybridoma technique and high-throughput sequencing
  • Antibody discovery method based on hybridoma technique and high-throughput sequencing
  • Antibody discovery method based on hybridoma technique and high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Isolation and Identification of Hybridoma Antibody Sequences

[0061] Use the following procedure:

[0062] (1) Expand and culture myeloma cells so that they are in the logarithmic growth phase when confluent. On the day of fusion, cells were collected, counted, and set aside.

[0063] (2) Take the hemagglutinin HA antigen polypeptide (hereinafter represented by the code GW01) and the immune checkpoint PD-L1 antigen polypeptide (hereinafter represented by the code GW-02) whose serum titer reaches the standard, and prepare hybridization with two immunized mice each Tumors were named GW01-1, GW01-3 and GW02-3, GW02-5, respectively. Eyeballs of the mice were removed to collect blood, and the serum was separated as a positive control serum for antibody detection. The mice were killed by pulling the neck, and the spleens of the mice were taken aseptically, the splenocytes were separated and the red blood cells were lysed to obtain pure B lymphocytes. Cell fusio...

Embodiment 2

[0066] Example 2 Preparation of antibody sequencing library and analysis of antibody sequences

[0067] The remaining spleen tissue of the hybridoma prepared in Example 1 and the bone marrow cells from the same immunized mouse were used to prepare the antibody sequencing library.

[0068] Specifically, the spleen tissue was ground in liquid nitrogen to extract total RNA, and the specific extraction procedure was carried out according to the manufacturer's instructions of the extraction reagent; the tibia and femur were collected from the same immunized mouse to remove muscle and fat tissue, and the Inject Trizol into the bone to wash out the bone marrow and collect it in a centrifuge tube to extract total RNA. The specific extraction procedure is carried out according to the manufacturer's instructions of the extraction reagent.

[0069] Using antibody variable region upstream primers and heavy chain and light chain variable region downstream primers, using the sample 5' RACE ...

Embodiment 3

[0092] Example 3 Comparison of Hybridoma and Antibody Variable Region Gene Profile Sequences and Selection of Candidate Matching Sequences

[0093] A total of 18 GW02 and 23 GW01 monoclonal hybridoma cell lines were analyzed, and the variable region of the hybridoma antibody gene was submitted for IgBlast to determine the information of CDR3H and CDR3L fragments. Comparing CDR3H and CDR3L with the corresponding antibody variable region gene profiles, most of the hybridoma CDR3H and CDR3L are included in the antibody variable region gene profiles, and the number of sequencing entries corresponding to some CDR3s exceeds 1% of the total number of sequencing entries. Belongs to high abundance CDR3. The number of high-abundance CDR3 and the proportion of hybridoma CDR3 in the antibody variable region gene profile are shown in Table 9 below.

[0094] Table 9

[0095]

[0096] In Table 9, CDR3>1%: the number of hybridoma CDR3s exceeding 1% of the number of antibody sequencing li...

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Abstract

The invention relates to a method for generating a recombinant antibody combining at least one target antigen and the recombinant antibody generated thereby. The antibody generation method provided by the invention combines an optimized high-throughput antibody sequencing method with a hybridoma antibody screening technology, and compared with the traditional hybridoma monoclonal antibody technology, enlarges the selection range of candidate antibody genes, improves the antibody structure optimization efficiency, and increases the possibility of acquiring functional antibodies.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a method for producing a recombinant antibody binding to at least one target antigen based on hybridoma technology and high-throughput sequencing, and the recombinant antibody obtained by the method. Background technique [0002] Hybridoma technology is the most commonly used antibody mining technology, but its steps are cumbersome, and the subcloning process required to obtain monoclonal cells is time-consuming and costly. [0003] In recent years, high-throughput sequencing technology has been increasingly applied to the characterization and analysis of variable region gene profiles of immune antibodies, as well as the discovery of antigen-specific antibodies. The method obtains the antibody variable region gene profile of the immunized animal through high-throughput sequencing of the immunoglobulin gene before and after immunization. Antibody genes that are signif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/10
CPCC07K16/1018C07K16/28C07K2317/55C07K2317/565C07K2317/622
Inventor 陈维之王晓红洪媛媛陈豫孙中平
Owner GENEWIZ INC SZ
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