A method for rapidly preparing an antigen-specific antibody

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

Active Publication Date: 2019-04-19
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 preparing an antigen-specific antibody
  • A method for rapidly preparing an antigen-specific antibody
  • A method for rapidly preparing an antigen-specific antibody

Examples

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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

A method for rapidly preparing an antigen-specific antibody is provided. The method includes establishing an antibody-polypeptide corresponding relationship and an antibody-motif corresponding relationship; finding a polypeptide having a same amino acid sequence with target protein by searching the antibody-polypeptide corresponding relationship, with an antibody corresponding to the polypeptide being the antigen-specific antibody; or calculating the similarity degree between all motifs of each antibody and all modules of the target protein in the antibody-motif corresponding relationship, with an antibody corresponding to a motif the similarity degree of which is greater than 0.4 being the antigen-specific antibody. A phage display technology and a high throughput sequencing technology are combined in the method, information of a polypeptide or motif that can be recognized by the given antibody can be efficiently acquired in an overall manner, the antibody-polypeptide corresponding relationship or the antibody-motif corresponding relationship is established, and through the relationship, the specific antibody corresponding to the target protein can be rapidly determined. The method has a wide application prospect.

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 Applications(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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