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Screening method of single memory B cell and application of single memory B cell in preparation of small-molecular monoclonal antibody

A monoclonal antibody and screening method technology, applied in the field of cell biology and immunology, can solve the problems of insufficient reverse transcription, easy volatilization and degradation, transcript loss, etc., and achieve high practical value, high affinity, and good application. Foreground effect

Active Publication Date: 2021-01-26
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibody preparation of traditional single B cells requires artificial single-cell RNA extraction and transcript amplification, but the amount of single-cell RNA is small, volatile and degradable, and has strict environmental requirements, and has undergone insufficient reverse transcription and low After efficient amplification, some important transcripts will be lost

Method used

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  • Screening method of single memory B cell and application of single memory B cell in preparation of small-molecular monoclonal antibody
  • Screening method of single memory B cell and application of single memory B cell in preparation of small-molecular monoclonal antibody
  • Screening method of single memory B cell and application of single memory B cell in preparation of small-molecular monoclonal antibody

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preparation example Construction

[0050]The preparation method of chloramphenicol immunogen CAP-KLH is as follows: 1 mg CAP-HS was dissolved in 500 μL DMF, 2.75 mg NHS was dissolved in 200 μL DMF, 2.75 mg EDC was dissolved in 400 μL DMF, the three were mixed, and incubated for 6 hours at room temperature with shaking. 500 μL of the reaction solution was slowly added dropwise to 2 mg of KLH, and the reaction was shaken at 4°C overnight. Another 500μL reaction solution was added with 2mg BSA, and dialyzed for 72h. The resulting coupling solution was aliquoted and stored at -20°C ( figure 1 ).

[0051] The preparation method of the immune preparation is as follows: CAP-KLH (concentration: 4 mg / mL) and complete Freund's adjuvant / incomplete Freund's adjuvant are mixed in equal volume and emulsified.

[0052] The way of immunization is as follows: multi-point injection immunization method on the back of the neck. The number of immunizations was 8 times. The first immunization was the primary immunization with Fre...

Embodiment 1

[0064] The immunization of embodiment 1 New Zealand white rabbit

[0065] The concentration of the immunogen (CAP-KLH) was adjusted to 4 mg / mL with PBS buffer, and the concentration of the immunogen was determined by the Bicinchoninic Acid method.

[0066] Take 6 New Zealand white rabbits (2 months old, body weight 2.5kg), numbered CAP-KLH-1, CAP-KLH-2, CAP-KLH-3, CAP-KLH-4, CAP-KLH-5 and CAP -KLH-6. New Zealand white rabbits were immunized eight times, and the way of immunization was 4-8 points on the back of the neck.

[0067] The first immunization: each mouse was immunized with 1 mL of the first immunization preparation (each 1 mL of the first immunization preparation contained 1 mg of CAP-KLH).

[0068] The second to eighth immunizations: Counting the days from the first immunization, one immunization was carried out every 30 days, and a total of 7 booster immunizations were carried out; the first 3 booster immunizations were immunized with 1 mg CAP-KLH each time; the l...

Embodiment 2

[0069] Embodiment 2 Affinity identification of New Zealand white rabbit antiserum

[0070] The titer and sensitivity of the antiserum obtained one week after immunization were tested. The assay method is indirect ELISA. The specific operation steps are as follows:

[0071] Dilute the coated CAP-BSA with carbonate buffer (pH9.6) to 0.1 μg / mL, coat the ELISA plate with an amount of 100 μL / well, incubate at 37°C for 2 hours, and then wash the plate 3 times with PBST solution. The ELISA plate was then blocked with 2% skimmed milk powder (150 μL / well), incubated at 37° C. for 1 h, washed three times with PBST solution, and patted dry. The antiserum was serially diluted with PBS, starting from a dilution of 1:4000, with a 2-fold gradient for a total of 8 dilutions. Add 50 μL of PBS buffer and 50 μL of serially diluted antiserum solution to the coated ELISA plate, incubate at 37°C for 30 min, then wash with PBST solution 3 times, and pat dry. Add 100 μL enzyme-labeled secondary a...

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Abstract

The invention provides a screening method of a single memory B cell and application of the single memory B cell in preparation of a small-molecular monoclonal antibody. The screening method is based on a fluorescence-activated cell sorting technology and a microfluidic technology, organic combination of accurate screening of an antigen-specific memory B cell and efficient preparation of a single cell transcript in a microenvironment is realized, and single cell variable region pre-amplification, scFv prokaryotic expression and eukaryotic expression of the monoclonal antibody are further carried out. A novel single cell antibody preparation technology is established, and a new means is provided for preparation of the small-molecular rabbit monoclonal antibody.

Description

technical field [0001] The present invention relates to the fields of cell biology and immunology, in particular to a method for screening single memory B cells and its application in the preparation of small molecule monoclonal antibodies. Background technique [0002] Rabbit monoclonal antibodies are widely used in the fields of biology, medicine, environmental testing and food science due to their advantages of high specificity, high activity and high affinity and recognition of more novel epitopes. With the development of monoclonal antibody technology, American Epitomics Company prepared rabbit myeloma cells, and successfully obtained rabbit monoclonal antibodies through hybridoma technology, but the instability of rabbit myeloma cell lines prevented the technology from being widely promoted use. [0003] Recombinant rabbit monoclonal antibodies have the advantages of high consistency and reproducibility on the basis of maintaining the advantages of the above-mentioned...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N15/10C07K16/44C07K16/06G01N33/577G01N33/53
CPCC12N5/0635C12N15/1096C07K16/44C07K16/06G01N33/577G01N33/5308C12N2509/00C07K2317/56C07K2317/92C07K2317/622C07K2317/94C12Q2531/113
Inventor 王战辉沈建忠温凯余文博于雪芝江海洋李园李培培
Owner CHINA AGRI UNIV
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