Preparation and application of human interleukin-6 receptor (hIL6R)-resistant antibody with high affinity
A kind of affinity and antibody technology, applied in the field of biomedicine, can solve the problems of large vacancies in the global market
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0015] Example 1, Preparation of hIL6R Mouse Monoclonal Antibody
[0016] 1. Preparation of hIL6R mouse monoclonal antibody
[0017] 1) Animal immunization: In order to obtain a mouse monoclonal antibody that can recognize hIL6R protein, the invention uses the recombinant hIL6R extracellular domain fragment produced by Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. as an immunogen to immunize Balb / c mice. Rats were immunized with 50 μg. For the first immunization, the immunogen and the same amount of complete Freund's adjuvant were used to make an emulsifier, and the abdomen was injected subcutaneously at multiple points, and the same dose of immunogen and the same amount of incomplete Freund's adjuvant were used to make an emulsifier at intervals of 3 weeks , boosted immunization twice, measured serum titer after three times of immunization, took mice with high serum titer and boosted intraperitoneal immunization once with immunogen, and took spleen 3 days later for fusion. ...
Embodiment 2
[0020] Example 2, Screening and identification of antibodies
[0021] After obtaining multiple strains of hIL6R mouse monoclonal antibodies, the antibodies were initially identified and screened, including the identification of antibody affinity and the detection of neutralizing activity:
[0022] Identification of antibody affinity: preliminary identification of antibody affinity was performed on the obtained mouse monoclonal antibody, and the affinity of the screened antibody was verified using Octet biomolecular interaction analyzer, and the hIL6R single Affinity determination of cloned antibodies. The material used therein is biotin-labeled recombinant hIL6R protein at a concentration of 30 μg / ml. After exploring the conditions, the antibody was diluted to 4 different concentrations, 3.33nM, 1.67nM, 0.8333nM and 0.4167nM. By comparing the affinity of each antibody, the antibody with better affinity is selected.
[0023] Antibody neutralization activity detection: Antibo...
Embodiment 3
[0031] Example 3, Obtaining of Monoclonal Antibody Genes
[0032] Hybridoma cells in good condition were collected, total RNA was extracted by adding TRIzol, the quality was detected by electrophoresis, and the concentration was determined by UV. The RNA was then reverse-transcribed into cDNA using a commercial kit, and frozen at -20°C for future use. According to references (Ying W et al., BMC Bioinformatics.2006; 7(Suppl 4): S9), primers were designed using technical schemes known to professionals in the field, and the light chain and heavy chain of the antibody were respectively obtained by PCR using cDNA as a template fragment. The light chain and heavy chain fragments were inserted into the pcDNA3T vector to construct pcDNA3-anti-hIL6R-L and pcDNA3-anti-hIL6R-H vectors. Transform Escherichia coli, pick positive clones, perform sequencing identification, analyze the sequencing results, and obtain the correct light chain and heavy chain amino acid sequences.
[0033] Det...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More