Monoclonal antibodies (mAbs) are a very valuable research tool for studying this diverse family of proteins and their interaction with human receptors[1]. Fusing mouse splenocytes with myeloma cells to generate long-lived antibody secreting hybridoma cell lines have been used since the 1970s for the production of mAbs. MAbs are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers[2]. The mouse is the most widely used animal for monoclonal antibody production because of its breeding capabilities and its highly efficient hybridoma production[3].
The screening of antigen-specific B cells has been pivotal for biotherapeutic development for over four decades. Conventional antibody discovery strategies, including hybridoma technology and single B cell screening, remain widely used based on their simplicity, accessibility, and proven track record[4]. Purifing recombinant protein was injected into BALB/c mice to perform an immunization procedure and stable positive monoclonal cell lines were screened and established. The sensitivity and specificity of the monoclonal antibodies against were determined using immunoblotting, immunoprecipitation and immunofluorescence analyses[5].
Bioscience has been committed to antibody preparation research for many years. We have experienced antibody preparation technicians, accumulated sufficient experience in antibody customization and recombinant protein production, and can provide customers with recombinant monoclonal antibody preparation services for different species, including rabbit, mouse, camel, sheep, chicken and shark. Relying on the antibody platform, protein platform and other perfect platform system construction, to provide one-stop technical services. Including antigen design, antibody modification, DNA synthesis, plasmid construction, protein expression, protein purification, protein modification, peptide synthesis, peptide modification, peptide and carrier protein cross-linking, small molecule structure analysis and carrier protein cross-linking, etc. If required, we also provide antibody purification and antibody identification services.
For other services please contact our customer service department.
website: www.biotyscience.com Phone: + 400-669-8850 Email: biotyscience@gmail.com
Antigen Preparation
Customers may provide highly purified (>90%) protein or peptide antigens that are ready for injection. Alternatively, we provide services for protein production, peptide design, peptide synthesis, and peptide conjugation to carrier protein for use as the immunogen.
Immune Animal
1.Balb/cfemale mice (4-6 weeks old) were selected. Hybridoma cells producing monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane.
2.After ten days, ascitic fluid was harvested from the peritoneum of each mouse.
Cell screening, Fusion and Subcloning
1.Spleen lymphocytes of immunized mice were fused with myeloma cells and screened to obtain monoclonal antibody specific cell lines.
2.Subcloning is important for the long-term stability of cloning and for ensuring that cells are truly monoclonal and maintain clonality throughout their productive lives. Positive clones were selected for subclonal expansion culture, and positive subclones were screened by ELISA for expanded culture. After fusion, single cloning by limited dilution method and supernatant detection, multiple cell lines meeting the project requirements were obtained, and the cell lines among them were selected for subsequent preparation of ascites.
Purification and Assay
1. Monoclonal antibodies are produced in cell culture in roller bottles to yield antibody-containing supernatants.
2.The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit.
3.MAb was purified from the ascitic fluid by ion exchange chromatography.
4.The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP.
Mouse monoclonal antibodies：optimized and efficient protocols for immunizing mice, creating fusions, screening hybridomas, and delivering cell lines
Customizable：you decide (based on our screening results and your own testing) which immunized mice to fuse and which cell lines to subclone and expand
Full-service integration：combine these protocols with our complete set of services for initial antigen preparation and subsequent antibody production and purification
Professional technical support：ensure strong, thoughtful and timely pre-sales and after-sales service support, at any time to answer your questions
Reference
[1]Marzieh R ,Abbas G .Production of a Mouse Monoclonal Antibody Against Mortalin by Whole Cell Immunization.[J].Monoclonal antibodies in immunodiagnosis and immunotherapy,2017,36(4):169-175.
[2]Ezzatifar F ,Majidi J ,Baradaran B , et al.Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice[J].Advanced Pharmaceutical Bulletin,2015,5(1):97-102.
[3]Tan Z ,Zhang J ,Su Z , et al.Production of rabbit monoclonal antibodies against mouse embryonic stem cells and identification of pluripotency-associated surface antigens[J].Journal of Immunological Methods,2011,365(1-2):149-157.
[4]Schardt S J ,Sivaneri S N ,Tessier M P .Monoclonal Antibody Generation Using Single B Cell Screening for Treating Infectious Diseases.[J].BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy,2024,
[5]Xiong W ,Huang W ,Jiao Y , et al.Production, purification and characterization of mouse monoclonal antibodies against human mitochondrial transcription termination factor 2 (MTERF2)[J].Protein Expression and Purification,2012,82(1):11-19.
Ordering Information
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Phone: +86-400-669-8850
Email: biotyscience@gmail.com