A method for rapidly obtaining antigen-specific antibodies

A specific, antibody technology, applied in chemical instruments and methods, biochemical equipment and methods, specific peptides, etc., can solve problems such as rapid acquisition of antigen-specific antibodies that are not involved or not involved, and achieve accelerated acquisition, accuracy and accuracy. Reliable effect

Active Publication Date: 2021-12-07
抗码(苏州)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Does not involve resolving peptides or proteins that interact with the antibody, nor does it involve the rapid acquisition of antigen-specific antibodies

Method used

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  • A method for rapidly obtaining antigen-specific antibodies
  • A method for rapidly obtaining antigen-specific antibodies
  • A method for rapidly obtaining antigen-specific antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Establishing an antibody-polypeptide relationship table

[0069] 1. Purchase Ph.D. from NEB Company TM -12phage display peptide library kit (New England Biolabs Ltd., Cat. No. E8110S);

[0070] 2. At 4°C, use 3% BSA-containing TBST-II (TBS solution containing 0.5% Tween 20, see Table 1 for its components) solution to block the 96-well PCR plate overnight, 200 μL per well;

[0071] Table 1

[0072]

[0073] 3. Add 0.4 μg of antibody to each well of the PCR plate. As a control, no antibody was added to one of the wells;

[0074] 4. Add 10 μL phage random peptide display library (NEB TM Ph.D-12), 100 times the diversity of the library;

[0075] Incubate overnight at 5.4°C;

[0076] 6. Add 10 μL of protein G magnetic beads (Thermo Fisher Scientific Inc., Cat. No. 10003D). Immunoprecipitation for 4 hours at 4°C;

[0077] 7. Use a 96-well magnetic stand to separate the magnetic beads. Wash 3 times with 200 μL TBST-II solution;

[0078] 8. Wash once wi...

Embodiment 2

[0135] Example 2, establishing antibody-motif relationship

[0136] 1. According to the requirements of the program MEME for the input file, input the polypeptide sequence corresponding to each antibody in the antibody-polypeptide relationship, calculate and output the 8 motifs, motif matrix and corresponding E value corresponding to the antibody, among which the The sequence matrix represents the contribution degree of each amino acid residue in the given position of the corresponding motif; the calculation of the motif matrix adopts a well-known method MEME.

[0137] 2. With 0.05 as the threshold, select motifs with E values ​​less than 0.05 and the corresponding motif matrix;

[0138] 3. Integrate all antibodies, E values ​​and motif matrixes to establish antibody-motif relationship tables (see Table 7 and Table 8).

[0139] Table 7 Antibody-Motif Relationship Table 1

[0140]

[0141]

[0142]

[0143] Table 8 Correspondence between antigens and motifs Table 2 ...

Embodiment 3

[0147] Example 3. Obtaining an antibody targeting protein SPA

[0148] 1. Using the polypeptide sequence in the antibody-polypeptide relationship table, the amino acid sequence SEQ ID No.33 corresponding to the antigen SPA (such as Figure 4 shown) comparison, it was found that a polypeptide ASWGSPHNRTSH recognized by the antibody Ab048 was completely identical to a part of the amino acid sequence in the antigen SPA, and the antibody Ab048 against the antigen SPA was obtained immediately;

[0149] 2. Use 10% separation gel, the loading volume is 10 μL, absorb 8 μL of protein SPA, and 2 μL of SDS-PAGE loading buffer, after mixing, denature at 95°C for 10 minutes. The sample loading volume was 10 μL, and 5 μL of molecular weight standards were added at the same time for SDS-PAGE electrophoresis. The electrophoresis conditions are 80V, 30min; 120V, 90min;

[0150] 3. Use cellulose acetate membrane. The membrane transfer condition is 160mA, 78min; (see Table 9 for the formulati...

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Abstract

The present invention provides a method for rapidly obtaining antigen-specific antibodies, including: establishing the corresponding relationship between antibodies and polypeptides and motifs; searching for the corresponding polypeptides with the amino acid sequence of the target protein by searching the corresponding relationships between antibodies and polypeptides, and the corresponding The antibody is an antigen-specific antibody; or calculate the similarity between all motifs of each antibody and all modules of the target protein in the corresponding relationship between the antibody and the motif, and select the motif corresponding to the similarity value greater than 0.4, The corresponding antibody is an antigen-specific antibody. The present invention combines phage display technology and high-throughput sequencing technology to efficiently and globally obtain information on polypeptides or motifs that can be specifically recognized by a given antibody, and establish a corresponding relationship between antibodies and polypeptides or motifs. Through the corresponding relationship, The specific antibody corresponding to the target protein can be quickly determined, and has very broad application prospects.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to a method for rapidly obtaining antigen-specific antibodies. Background technique [0002] Antibodies are one of the most important reagents and drugs in life sciences, medicine, pharmacy and other related fields, and play an irreplaceable role. Antibodies are currently mainly obtained by immunizing animals. Antibodies include monoclonal antibodies with single antibody components, engineered antibodies, and modified antibodies, as well as polyclonal antibodies with complex antibody components. Among them, the complex composition and low reproducibility of polyclonal antibodies limit its application range. At present, the main ways to obtain single-component antibodies are hybridoma technology (Nature 1975, 256, 495-497); biopanning antibody display library (Nature Biotechnology 1996, 14, 309-314.), etc., but these technologies are time-consuming and laborious, and it is diffi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00G01N33/53
CPCC07K16/005C12Q1/6869G01N33/53C12Q2535/122C12Q2537/143C12Q2531/113
Inventor 陶生策祁环赵小东李华胡传圣
Owner 抗码(苏州)生物科技有限公司
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