Single-domain antibody resistant to human beta2-microglobulin as well as preparation method and application of single-domain antibody

A β2 microglobulin and single domain antibody technology, applied in the field of anti-β2 microglobulin antibodies, can solve the problems of increased sensitivity, high production cost, limited antibody immobilization density, etc., to promote diagnosis and treatment, high specificity Binding capacity, improved diagnosis and treatment outcomes

Active Publication Date: 2014-10-15
CROWN MEDICAL TECH DALIAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of IgG in the field of β2 microglobulin detection has the following defects: (1) the larger molecular weight limits the immobilization density of antibodies on the surface of the matrix, and also increases the difficulty of directional immobilization of antibodies, which limits the improvement of the sensitivity of the above detection methods
(2) Monoclonal IgG is difficult to prepare in large quantities, an

Method used

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  • Single-domain antibody resistant to human beta2-microglobulin as well as preparation method and application of single-domain antibody
  • Single-domain antibody resistant to human beta2-microglobulin as well as preparation method and application of single-domain antibody
  • Single-domain antibody resistant to human beta2-microglobulin as well as preparation method and application of single-domain antibody

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0037] Example 1 Screening of monoclonal phage antibodies

[0038] Using phage display technology to carry out multiple rounds of enrichment and screening of the alpaca phage antibody library to purify β 2 Microglobulin has undergone four rounds of enrichment screening of "adsorption-elution-amplification" for the antigen, showing that the recovery rate of each round of screening is increasing, indicating that after screening, it has β 2 Microglobulin-specific phages are highly enriched.

[0039] The specific method is as follows:

[0040] 1. Establishment of alpaca phage antibody library

[0041] The names of the primers and their sequences are shown in Table 1. The underlined part is the restriction site.

[0042] Table 1. Primer name and its sequence

[0043]

[0044]

[0045] (1) Separation of lymphocytes and separation of total RNA

[0046] Take 100ml of peripheral blood from a 3-year-old female alpaca, put it into an anticoagulation tube, and add an equal volume of lymphocyte separa...

Example Embodiment

[0084] Example 2 Detection of monoclonal phage antibodies:

[0085] (1) Randomly pick 80 single bacterial clones from the TY-AG plate cultured overnight after the fourth round of screening. After mixing BLT5615 with the phage preservation solution / lysate (1‰ to 0.1‰), culture them on a shaker at 37°C. After lysing the bacteria, add NaCl to the concentration of 0.5M in the solution immediately, centrifuge at 10000rpm for 10min, take the supernatant, and recover the phage solution. ELISA method for detection: Antigen β 2 The microglobulin was coated with an ELISA plate (0.4μg / well), 100μl of monoclonal phage solution was added to each well after sealing, and incubated at 37°C for 2h. After washing, add 1:5000 HRP-labeled mouse anti-M13 antibody, and incubate at 37°C for 1 hour. The positive control is the primary antibody (mouse anti-human β 2 Microglobulin), secondary antibody (goat anti-mouse IgG); negative control only added 2% MPBS. Using o-phenylenediamine as the substrate, ...

Example Embodiment

[0087] Example 3 Determination of DNA sequence of positive clones:

[0088] The phagemids of A and B positive clones were extracted respectively, using sdAb DNA sequencing primers (upstream primers and downstream primers from T7select cloning kit), the SdAb DNA sequence was determined by dideoxy end termination method.

[0089] Obtain the nucleotide sequences of clones A and B, and use Blast software to perform a comprehensive comparison and analysis of the obtained sequences with the single domain antibody variable region genes in Gene Bank. The analysis results show that the variable region genes of clones A and B are in line with the SdAb antibody gene, and the deduced amino acid sequence has a typical antibody variable region structure. The protein sequence determines the start and end of the CDR according to the blast and Kabat codes. Location. Complementarity determining region (CDR) is highly variable in amino acid composition and arrangement. The three CDRs together const...

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Abstract

The invention discloses a novel single-domain antibody resistant to human beta2-microglobulin as well as a preparation method and an application of the single-domain antibody. A phage display technology is adopted to perform multiple rounds of screening on an alpaca phage antibody library to obtain phage enriched with beta2-microglobulin specificity, the phage is cultured for preparing the antibody, positive clone is obtained through identification, a coding sequence corresponding to the positive clone is obtained through sequencing, and then expression is performed in escherichia coli to obtain a soluble antibody fragment. The antibody has an amino acid sequence represented as SEQ ID NO.1 or SEQ ID NO.10. The antibody provided by the invention has very high bonding capacity for beta2-microglobulin, can be applied to the fields of blood purification and beta2-microglobulin detection, and facilitates promotion and improvement of diagnosis and treatment for diseases such as dialysis-related amyloidosis and the like.

Description

technical field [0001] The present invention relates to an anti-β2 microglobulin antibody, in particular to a novel anti-β2 microglobulin specific single-domain antibody, the amino acid sequence and DNA coding sequence of its variable region, and a method for preparing the single-domain antibody and uses. Background technique [0002] β2 microglobulin is a small molecular globulin produced by lymphocytes, platelets, and polymorphonuclear leukocytes, with a molecular mass of 11.8 kDa. It is a single-chain polypeptide), and widely exists in plasma, urine, cerebrospinal fluid, saliva and colostrum, and the content is very low. In healthy people, β2 microglobulin is synthesized at a relatively stable rate, and the serum concentration of healthy people is relatively stable, generally 1.5-3mg / l (Tilman B, et.al, Massy beta2-microglobulin progress in uremic toxin research, Seminars in dialysis200922,4,378–380). The synthesis rate of β2 microglobulin in healthy people is about 2....

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/70C12N1/21G01N33/68
Inventor 贾凌云暴晓博徐丽任军
Owner CROWN MEDICAL TECH DALIAN CO LTD
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