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Directional coupling method based on recombinant genetic engineering antibody and microspheres and application

A genetically engineered antibody and gene recombination technology, applied in the field of immunodetection, can solve the problems of difficult to control the enzyme digestion process, complex process, no glycosylation modification, etc., to achieve excellent batch-to-batch consistency, improve accuracy, improve The effect of sensitivity

Active Publication Date: 2021-09-17
NANJING LEADING BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] CN112630420A, using the glycosylation site of the antibody to achieve, but this method has great limitations, most antibodies may not have glycosylation modification, or even if there is glycosylation modification, it may also be located at F(ab) ' end, so can not achieve directional coupling
[0009] CN107764992A, cut off the Fc segment of the antibody by enzyme, then open the disulfide bond in the hinge region of the antibody, and then realize directional coupling. This method also has relatively large limitations. In the process of reducing the disulfide bond, the F(ab)' disulfide bond may also be reduced, making the entire antibody lose specificity
The limitation of this method is that the operation steps are relatively complicated, and two proteins need to be linked successively on the microspheres, and the Fc end of the antibody has two chains, because it is easy to form agglutination when the Protein A fragment is combined, and the production process is relatively high.

Method used

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  • Directional coupling method based on recombinant genetic engineering antibody and microspheres and application
  • Directional coupling method based on recombinant genetic engineering antibody and microspheres and application
  • Directional coupling method based on recombinant genetic engineering antibody and microspheres and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of Genetically Engineered Thiolated Antibody

[0039] (1) After immunizing Balb / c mice with the recombinant target protein several times, the spleen was taken to separate lymphocytes; total RNA was extracted from the isolated lymphocytes with an RNA extraction kit, and synthesized by reverse transcription with a reverse transcription kit cDNA, the heavy chain variable region and light chain variable region genes were amplified with murine single-chain antibody scfv universal degenerate primers; the target gene was cloned into the phage vector pCANTAB5E (Pharmacia), and electroporated several times with a bacterial electroporation instrument Transform into Escherichia coli TG1 competent for electroporation, spread on 2× YTAG plate containing ampicillin resistance (50 μg / mL) and 2% glucose, cultivate overnight at 30°C, take an appropriate amount of 2× YT medium for use Scrape off all the colonies on the plate with a sterile glass rod and collect the ...

Embodiment 2

[0044] Example 2 Directional coupling of genetically engineered thiolated antibody to amino microspheres using a bifunctional cross-linking agent

[0045] combine figure 1 As shown, the specific experimental process is:

[0046] (1) Take Spherotech aminopolystyrene microspheres (AP-025-100) and dilute to 3mg / ml with PB, pH 7.2 buffer;

[0047] (2) Weigh 2 mg of SMCC and dissolve it with DMSO to 3 mg / ml; put the diluted SMCC solution into the above microsphere diluent at a volume ratio of 1:4, incubate at 37°C for 0.5 hours, and then centrifuge Remove the supernatant and resuspend with PB, pH7.2 buffer;

[0048] (3) Dilute the genetically engineered thiolated antibody prepared in Example 1 to 1 mg / ml with PB, pH 7.2 buffer solution, and add it to the above-mentioned SMCC-activated microspheres at a volume ratio of 1:5; 37°C Incubate with constant temperature and vibration for 1 hour, and add 1% bovine serum albumin solution to block after completion, and incubate with consta...

Embodiment 3

[0055] Example 3 Preparation of Serum Amyloid A Detection Reagent by Using the Directional Coupling Technology in Example 2 (Latex Enhanced Immunoturbidimetric Method)

[0056] Referring to the recombinant genetic engineering method of Example 1, two strains of paired thiolated anti-human SAA monoclonal antibodies were prepared, and antibody microsphere complexes were prepared respectively with reference to the method of Example 2. After the preparation was completed, the two antibody microsphere complexes 1: 1 Mix well to get the R2 reagent.

[0057] The specific components of the serum amyloid A detection reagent are as follows:

[0058] Reagent R1:

[0059] Na 2 HPO 4 12H 2 O 2.9 g / L

[0060] K H 2 PO 4 0.2 g / L

[0061] KCl 0.2 g / L

[0062] NaCl 8g / L

[0063] Tween 20 0.1% v / v

[0064] PEG 6000 10g / L

[0065] Proclin 300 0.1% v / v

[0066] Reagent R2:

[0067] Tris-HCl (pH8.0) 10 mM

[0068] Recombinant anti-human SAA antibody microsphere complex 3 g / L

[00...

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Abstract

The invention relates to the technical field of immunodetection, in particular to a directional coupling method based on a recombinant genetic engineering antibody and microspheres and application. The method comprises the following steps: A, carrying out gene recombination on a Fab segment of a mouse monoclonal antibody and an Fc segment of humanized IgG1, mutating the Fc segment of humanized IgG1, and introducing cysteine to the tail end of the Fc segment; and B, directionally coupling the Fc segment of the antibody to the amino microspheres through a bifunctional cross-linking agent. According to the present invention, the sulfydryl is accurately introduced into the Fc segment through the gene engineering antibody recombination manner, such that the complex pre-treatment step on the antibody during the reagent preparation can be avoided, and the batch-to-batch consistency of the reagent can be ensured; meanwhile, the humanized transformation is carried out on the Fc segment, so that interference of heterotropism antibodies and the like during sample detection can be avoided; in addition, by virtue of a directional coupling technology, the accuracy and sensitivity of reagent testing are improved.

Description

technical field [0001] The invention relates to the technical field of immunodetection, and specifically relates to a directional coupling method based on recombinant genetic engineering antibodies and microspheres. Background technique [0002] As a heterologous protein, mouse monoclonal antibody has many interference factors in the clinical detection process, and the detection interference caused by different regions and races is very large. Although monoclonal antibodies are widely used in many fields, according to the different diagnostic needs of the market, there are still many technical problems in obtaining human monoclonal antibodies through hybridoma technology, which cannot be processed and modified in the later stage, and the application of treatment and diagnosis and detection is obviously lagging behind. [0003] In the 1990s, the antibody library technology appeared, which bypassed the hybridoma pathway necessary for the development of monoclonal antibodies, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/546G01N33/74G01N33/92G01N33/68
CPCG01N33/531G01N33/54313G01N33/74G01N33/92G01N33/86G01N2333/775G01N2333/585G01N2333/75
Inventor 张玉基王鹏顾佳王倩仲子进夏昌校
Owner NANJING LEADING BIOMEDICAL TECH CO LTD
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