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Method and system for screening specific BCR/TCR

A specific and sequence-based technology, applied in the fields of molecular biology and cellular immunology, can solve problems such as failure, multi-identification, and difficulty in screening human antibodies, and achieve the effects of improving efficiency, high specificity, and accuracy

Active Publication Date: 2021-07-16
CHENGDU EXAB BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The disadvantages of monoclonal technology are twofold: on the one hand, the number of BCR or TCR on the surface of a single lymphocyte is limited, which limits the sensitivity and specificity of single cell sorting by antigen, and the sorted cells cannot be guaranteed to be positive cells. In the later stage, more identification of the obtained antibody / TCR is still required; on the other hand, the difficulty of single cell cloning is relatively high, and there are high requirements on the quality of cells, the skills of operators, and the quality of reagents
[0012] There are still three significant deficiencies in the existing monoclonal antibody screening technology: the first is that it is difficult to screen human antibodies, because it is impossible to repeatedly immunize the human body with antigens and adjuvants like immunized animals
[0013] The second is the difficulty in preparing monoclonal antibodies against complex antigens
When it is necessary to prepare antibodies against larger and more complex antigens (such as membrane protein complexes, tumor cells, etc.), it is difficult to select and prepare antigens for immunization and there is a risk of failure, and the function cannot be guaranteed after immunization Antibodies that meet the above requirements (such as antibodies obtained by immunizing with polypeptide fragments, not necessarily neutralizing antibodies)
If multiple antigens are selected for immunization and screening at the same time, the cost of monoclonal antibody preparation will be significantly increased

Method used

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  • Method and system for screening specific BCR/TCR
  • Method and system for screening specific BCR/TCR
  • Method and system for screening specific BCR/TCR

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Example 1 Obtaining the Characteristic Antibody Heavy Chain Sequence Set in Peripheral Blood After Novel Coronavirus (COVID-19) Infection by High-throughput Sequencing

[0057] 1. Collect the peripheral blood of 1377 control groups (including healthy people and patients not infected with the new crown) and 94 patients infected with the new coronavirus:

[0058] (1) Use EDTA sterile blood collection tube (do not use heparin or citric acid), collect blood 0.5-1mL, flick the blood collection tube to mix the blood and anticoagulant evenly. If the collected blood is too late to be processed, it can be temporarily stored in a 4°C refrigerator (no more than 2 hours).

[0059] (2) Erect the blood collection tube, use a brand new disposable syringe, and inject 5mL TRIzol (TRIsol, RNAiso plus, SuperfecTRI all can) into the blood collection tube through the rubber stopper (just pass it, do not open the cap of the blood collection tube) . If the blood has stratified after being s...

Embodiment 2

[0125] Example 2. Screening of the most potential novel coronavirus antibody heavy chain sequence from the characteristic antibody heavy chain sequences in peripheral blood after novel coronavirus infection

[0126] 1. From the novel coronavirus characteristic antibody heavy chain sequence set, remove all antibody heavy chain sequences that appear in less than 10 new coronavirus infected samples (<10% of the number of new coronavirus infected patients). A total of 77 antibody CDR3 sequences were selected in this step.

[0127] 2. Sort the antibody heavy chain sequences screened in step 1 according to "the sum of the number of repeated occurrences of any unique antibody heavy chain CDR3 sequence in all novel coronavirus infection patient samples" from high to low, and select the top 100 Or the top 10%. Since there are few sequences selected in step 1, all of them are reserved in this step.

[0128] 3. According to the characteristics of the antigen protein, screen the antibod...

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Abstract

The invention discloses a method and a system for screening specific BCR / TCR. The method for screening the specific BCR / TCR comprises the following steps of (1) extracting mRNA in lymphocytes of an immunized sample to be detected; (2) carrying out reverse transcription treatment on the mRNA obtained in the step (1), then carrying out multiple PCR amplification on the BCR / TCR variable region gene, then carrying out high-throughput sequencing, and determining nucleic acid and amino acid sequences of the BCR / TCR variable region; and (3) comparing the sequence obtained by sequencing with a lymphocyte BCR / TCR sequencing result of a sample which is not stimulated by the antigen to obtain a corresponding specific immune sequence. The method disclosed by the invention has the advantages that by comparing big data of the immune spectrum and comparing a huge number of BCR / TCR sequences, the specificity and the accuracy are higher, the efficiency of screening and identifying the antibody and the T cell receptor can be greatly improved, and the screening cost is low.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and cellular immunology, and in particular relates to a method and system for screening specific antibodies (BCR) and T cell receptors (TCR). Background technique [0002] Monoclonal antibody is a highly uniform antibody that recognizes a specific epitope produced by a single B cell. Monoclonal antibodies have broad application prospects in basic medical and biological research, as well as in the diagnosis, prevention and treatment of clinical diseases. To obtain monoclonal antibodies against specific antigens, antibody screening is required. The existing relatively mature monoclonal antibody screening technologies include: [0003] 1. Hybridoma technology. [0004] The mouse splenocytes after antigen immunization are fused with mouse myeloma cells, and the hybrid cells formed can not only produce antibodies, but also proliferate indefinitely. The fused hybrid cells are divided into...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/6869G16B20/30G16B30/10G16B40/10
CPCC12Q1/686C12Q1/6869G16B30/10G16B20/30G16B40/10C12Q2537/143C12Q2535/122Y02A50/30
Inventor 张志新卓越杨鑫
Owner CHENGDU EXAB BIOTECH CO LTD
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