Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method of non-fusion anti-desmoglein DSG3 rabbit-derived monoclonal antibody

A technology of desmoglein and monoclonal antibody, which is applied in the field of monoclonal antibody preparation, can solve the problems of lack of industrial reproducibility, restrictions on the industrial production and application of antibodies, and the inability to carry out antibodies.

Pending Publication Date: 2020-09-01
湖南远泰生物技术有限公司
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The screening process of these two methods for preparing antibodies is extremely random and uncertain. Therefore, if the fusion cell line of the antibody cannot be obtained, the preparation of the antibody cannot be carried out, and it does not have industrial reproducibility. nature, which seriously restricts the industrial production and application of the antibody

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method of non-fusion anti-desmoglein DSG3 rabbit-derived monoclonal antibody
  • Preparation method of non-fusion anti-desmoglein DSG3 rabbit-derived monoclonal antibody
  • Preparation method of non-fusion anti-desmoglein DSG3 rabbit-derived monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] This example provides a specific preparation method of non-fused anti-desmoglein DSG3 rabbit-derived monoclonal antibody, specifically as follows:

[0064] 1. Immunization and blood test of DSG3

[0065] The immunized animals are healthy male New Zealand white rabbits aged about three months and weighing about 2.5kg, and the animals are immunized with special antigens. Specific steps are as follows:

[0066] Before the first immunization, blood was taken from the ear vein as a negative control. Take 500 μg of antigen, then add an equal volume of Freund’s complete adjuvant (after mixing and emulsification) for the first immunization, and mix an equal volume of 250 μg of antigen with Freund’s incomplete adjuvant (after mixing and emulsification) for the second to fourth Immunization. Rabbits were injected subcutaneously at limbs, underarms and back at multiple points, and the second immunization was performed three weeks after the first immunization, and a booster was ...

Embodiment 2

[0086] In this example, using the antibody preparation method described in the above example, two monoclonal antibodies of DSG3 rabbit origin were obtained: antibody 1C2 and antibody 1A4, against these two antibodies and coated DSG3, as well as irrelevant protein 1 and irrelevant protein 2 The antigen-coated plate was subjected to indirect enzyme-linked immunoreaction (ELISA method) to verify its specificity results.

[0087] The DSG3 rabbit-derived monoclonal antibody was diluted to different ratios for testing, and the results were as follows: figure 1 , figure 2 shown. The results showed that after antibody 2 3 After one-fold dilution, the binding responses of DSG3 rabbit monoclonal antibodies 1C2 and 1A4 to DSG3 were significantly different from those of unrelated proteins, and this difference existed until antibody 2 8 in the dilution results. It shows that both antibody 1C2 and antibody 1A4 only have specific adsorption reaction to DSG3 antigen.

Embodiment 3

[0089] In this example, a histoimmunochemical verification experiment was carried out for the prepared DSG3 rabbit-derived monoclonal antibodies 1C2 and 1A4, as follows:

[0090] The DSG3 rabbit monoclonal antibody 1C2, 1A4 was detected by immunohistochemistry under the gradient, and the positive serum was used as the antibody control, and the HRP color development results were as follows: image 3 shown.

[0091] Experimental conclusion, the immunohistochemical detection trend of DSG3 rabbit monoclonal antibody 1C2 and 1A4 in cervical cancer, skin cancer, colon cancer and gastric cancer is consistent with the positive serum, and the expression of DSG3 antigen protein is higher in cervical cancer and skin cancer expression in colon and gastric cancer.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a preparation method of a non-fusion anti-desmoglein DSG3 rabbit-derived monoclonal antibody. The preparation method comprises the following steps: 1) selecting a suitable immune animal, and carrying out DSG3 immunization on the animal; 2) extracting successfully immunized animal blood, collecting peripheral blood mononuclear cells, and separating out a B lymphocyte population capable of secreting a DSG3 antibody; 3) establishing a rabbit-derived single-chain antibody yeast display library; 4) constructing a DSG3 rabbit-derived monoclonal antibody expression vector library; 5) constructing a DSG3 rabbit-derived monoclonal antibody yeast expression library: namely converting a pichia pastoris expression vector PZ recombinant plasmid into yeast expression bacteria X33, and screening X33 recombinant transformation monoclonal bacteria capable of secreting a DSG3 antibody; 6) obtaining a X33 recombinant conversion monoclonal strain that can stably secrete a DSG3 antibody; and 7) purifying the obtained DSG3 rabbit-derived monoclonal antibody. The provided preparation method can prepare a stable DSG3 antibody, has industrial repeatability and is suitable for industrial popularization and application.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibody preparation, in particular to a preparation method of a non-fused anti-desmoglein DSG3 rabbit monoclonal antibody. Background technique [0002] Desmoglein (DSG) is a member of the cadherin superfamily, and the desmosomes composed of DSG play an important role in the process of cell-cell adhesion. At present, four DSGs have been discovered, among which DSG1 and DSG3 are closely related to dermatology, and are the targets of various autoimmune diseases including pemphigus and some hereditary bullous diseases. At the same time, more and more studies have shown that DSG may play an important role in the process of cell proliferation, apoptosis and migration, so it may be clinically relevant to the occurrence and development of squamous cell carcinoma. At present, the detection of serum anti-desmoglein DSG3 antibody has been used in the diagnosis of pemphigus, and plays a key role in the ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/06C12N15/81C12N1/19C12R1/84
CPCC07K16/06C07K16/18C07K2317/14C07K2317/92C12N15/815
Inventor 熊丹
Owner 湖南远泰生物技术有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com