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Antigen polypeptides and monoclonal antibodies that can be used to enrich human l1cam protein

A monoclonal antibody and antigenic polypeptide technology, applied in animal/human protein, anti-animal/human immunoglobulin, immunoglobulin, etc., can solve the problems of complicated monoclonal antibody screening methods, difficult screening, and high cost, and achieve Ingenious design, low cost, easy to obtain effects

Active Publication Date: 2021-01-22
SICHUAN BAILI PHARM CO LTD
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  • Description
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AI Technical Summary

Problems solved by technology

[0002] The existing antibody that can enrich human L1CAM (neural cell adhesion molecule L1) protein is derived from 16-week-old human fetal brain suspension. For general antibody production and R&D companies, it is very difficult to obtain the same source of antigen. It is difficult, and after immunization with human fetal brain suspension at about 16 weeks, the screening method of the later monoclonal antibody is more complicated, and the screening is very difficult and costly

Method used

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  • Antigen polypeptides and monoclonal antibodies that can be used to enrich human l1cam protein
  • Antigen polypeptides and monoclonal antibodies that can be used to enrich human l1cam protein
  • Antigen polypeptides and monoclonal antibodies that can be used to enrich human l1cam protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Antigenic epitope analysis of L1CAM protein: Using DNAStar software, by analyzing the antigenicity, surface exposure, hydrophilicity, and flexibility of each segment of the protein, the 1071-1086 amino acid REGSQRKHSKRHIHKD and the 854-869 amino acid REGSQRKHSKRHIHKD of L1CAM were determined. Each segment has 16 amino acids as a synthetic amino acid sequence. In order to facilitate coupling to the carrier, cysteine ​​(Cys) is added to the N-terminal of the two polypeptides to obtain (C) NQSSYTQWDLQPDTDY, (C) REGSQRKHSKRHIHKD according to the initial and last letters of the polypeptide And the number of polypeptides were named CY-17 and CD-17, respectively.

[0034] The synthesis of peptides is synthesized and purified by an automatic synthesizer; the two-stage synthesized peptides are coupled to the carrier protein KLH using the EDC coupling method to form CY-17-KLH and CD-17-KLH conjugates, and then at pH 7.4 Dialyze the medium in PBS.

[0035] Antibody preparation me...

Embodiment 2

[0041] Using DNAStar software to analyze the antigenicity, surface exposure, hydrophilicity, and flexibility of the relevant regions of the L1CAM extracellular domain protein sequence, and determine the 1071-1086 amino acid REGSQRKHSKRHIHKD and the 854-869 amino acid REGSQRKHSKRHIHKD of L1CAM, each with 16 segments amino acid as a synthetic amino acid sequence, with Figure 1 shown.

[0042] Collect mouse serum after immunization, carry out titer detection, the result is as follows:

[0043]

[0044] Screening of monoclonal antibody cell lines with biological activity, and detection of biological activity with cell culture supernatant:

[0045] Detection 1, WB, loading sample: mouse brain tissue column solution, primary antibody is cell culture supernatant, secondary antibody: goat anti-mouse-HRP1:5000, the position of the arrow indicates the position of the target band.

[0046]Detection 2, IP-WB, loading sample: L1CAM of human serum enriched in cell culture supernatant,...

Embodiment 3

[0052] 1. Use ProteinA beads to absorb anti-L1CAM IgG:

[0053] A in the immune serum detection stage: use ProteinA beads to absorb a small amount (5ul) of immune mouse serum antibody for 4h to form a Protein A-IgG complex (here Protein A only binds to IgG antibodies, including IgG stimulated by our antigen, but does not bind to serum other protein components in).

[0054] B In the cell line screening stage, use ProteinA beads to adsorb the cell culture supernatant to form a Protein A-IgG complex. Since the IgG concentration of the culture supernatant in the culture supernatant is very low, we use ProteinA beads to adsorb 1ml of the cell culture supernatant .

[0055] C In the antibody verification stage, we use ProteinA beads to adsorb 5ug of antibody.

[0056] At each stage, we performed positive control experiments in the process of using ProteinA beads to adsorb anti-L1CAM IgG. The anti-L1CAM antibody from abcam ([UJ127](ab3200)) was used as a positive control antibody t...

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Abstract

The invention discloses antigen polypeptide and a monoclonal antibody applicable to enrichment of human L1CAM protein. Through adoption of polypeptide antigen immunization design and ingenious antibody screening design, antibodies obtained from a brain suspension immunogen of an about 16-week-old human fetus and having the same human L1CAM protein enriching functional activity are obtained in a very simple way, and some antibodies can also be applied to biological functions of recognizing various protein forms of human L1CAM in a WB way.

Description

technical field [0001] The invention relates to an antigen polypeptide and monoclonal antibody that can be used to enrich human L1CAM protein. Background technique [0002] The existing antibody that can enrich human L1CAM (neural cell adhesion molecule L1) protein is derived from 16-week-old human fetal brain suspension. For general antibody production and R&D companies, it is very difficult to obtain the same source of antigen. It is difficult, and after immunization with human fetal brain suspension at about 16 weeks, the screening method of the later monoclonal antibody is more complicated, and the screening is very difficult and costly. Contents of the invention [0003] The purpose of the present invention is to provide an antigen polypeptide and monoclonal antibody that can be used to enrich human L1CAM protein. [0004] In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problems is: [0005] An ant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C07K16/28
CPCC07K14/70503C07K16/2803
Inventor 朱义张勇卓识陈澜
Owner SICHUAN BAILI PHARM CO LTD
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