Method for sorting and screening nano antibody through flow cytometry

A nanobody, sorting and screening technology, used in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc. The effect of avoiding phage contamination, less workload, and shorter screening cycle

Pending Publication Date: 2018-07-24
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the steps of RNA extraction and reverse transcription, and RNA is a substance that is easily contaminated and degraded, it is difficult to ensure a high enough screening library capacity,

Method used

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  • Method for sorting and screening nano antibody through flow cytometry
  • Method for sorting and screening nano antibody through flow cytometry
  • Method for sorting and screening nano antibody through flow cytometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Using Flow Cytometry to Screen Anti-Pseudomonas Exotoxin A Nanobodies

[0052] figure 1 A preferred embodiment of the method disclosed in the present invention is given in figure 1 Firstly, B cells with unlabeled antibody were used for flow cytometry detection, and this was used as a negative control; at the same time, Anti-llama pan B cell monoclonalantibody was used as the primary antibody, Rat Anti-Mouse IgM-PE was used as the secondary antibody to label B cells, and FITC was used to label B cells Pseudomonas aeruginosa exotoxin A; establish the FSC-H / FSC-W experimental plate, and finally obtain PE and FITC double positive samples.

[0053] 1.1 Antigen-immunized alpaca: A healthy adult alpaca was selected, and the peripheral blood of the alpaca was collected as a negative control not immunized with antigen, and stored in a -80°C ultra-low temperature refrigerator. Mix the recombinant Pseudomonas aeruginosa exotoxin A as antigen with Freund's adjuvant at a...

Embodiment 2

[0099] Example 2. Analysis of affinity activity between nanobody and antigen Pseudomonas aeruginosa exotoxin A

[0100] 2.1 Chip antigen coupling: Prepare the antigen with different pH sodium acetate buffer (pH 5.5, pH 5.0, pH 4.5, pH 4.0) to prepare 20 μg / mL working solution, and prepare 50 mM NaOH regeneration solution at the same time, use Biacore The template method in the instrument of the T100 protein interaction analysis system analyzes the electrostatic binding between antigens at different pH conditions and the surface of the chip (GE Company). Use a neutral pH system and adjust the antigen concentration as needed for coupling conditions. The chip was coupled according to the template method that comes with the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the designed theoretical coupling amount. The coupling process takes about 60 min.

[0101] 2.2 Exploration of analyte concentration...

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Abstract

The invention discloses a method for sorting and screening nano antibody through flow cytometry. The method comprises the following steps: (1) collecting peripheral blood B lymphocyte of a camelidae animal with immune target antigen; (2) sorting the B lymphocyte as a single cell by applying target antigen through the flow cytometry; (3) directly performing reverse transcriptional reaction on the sorted single B lymphocyte to generate cDNA; (4) taking the cDNA as a template, performing PCR amplification on an antibody heavy chain sequence and recycling an amplified product; (5) taking the amplified product in the step (4) as a template, and performing PCR amplification on CH1 and CH2 sequence coding genes of the antibody; (6) taking the amplified product in the step (4) which is amplified to be negative in the step (5) as a template, and performing PCR amplification on a VHH segment coding gene of the antibody; (7) cloning a VHH segment obtained in the step (6) into an expression vector, and expressing the VHH segment in host bacteria; and (8) identifying the nano antibody which is pressed in the step (7). The method disclosed by the invention is simple to operate, is short in screening period, avoids phage pollution risk, improves screening efficiency, reduces workload greatly, is low in economic cost, and is suitable for industrial large-batch screening requirements.

Description

technical field [0001] The invention discloses a method for screening nanobodies. Background technique [0002] In 1993, Hamers-Casterman et al. found that a class of only heavy chain dimer antibody H was found in camelids (camel, dromedary and llama). 2 , which are mainly IgG2 and IgG3 types. Such antibodies are called heavy chain-only-like antibodies (HCAbs) due to the lack of light chains, and their antigen-binding sites are composed of a domain called the VHH region, so Such antibodies are also referred to as single domain antibodies or single domain antibodies (sdAbs). Because this type of antibody is the variable region sequence after the constant region is removed, the molecular weight is only 15kD, and the diameter is about 10 nanometers, so it is also called nanobody (Nbs). Additionally, this class of single-domain antibodies, known as VNARs, has also been observed in sharks. This heavy-chain-only antibody was originally recognized only as a pathological form of...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K16/12C12N15/13G01N15/14
CPCC07K16/1214C07K2317/569C07K2317/565G01N15/1404
Inventor 宋海鹏刘原源于建立周宇杭李飞古一
Owner 深圳市国创纳米抗体技术有限公司
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