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Published on: June 5, 2015
Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen
1Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
Insights
Researchers developed a novel method to create monoclonal antibodies (MAbs) targeting the CD14 molecule, crucial for identifying monocytes and understanding endotoxin responses.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- CD14 is a leukocyte surface molecule primarily on monocytes.
- CD14 acts as a receptor for bacterial endotoxin.
- Monoclonal antibodies (MAbs) against CD14 are valuable for cell identification and studying immune responses.
Purpose of the Study:
- To develop an efficient method for producing CD14-specific MAbs.
- To utilize a novel antigen preparation strategy for MAb generation.
- To characterize the specificity and function of newly generated CD14 MAbs.
Main Methods:
- Generated CD14-expressing COS cells using cDNA and expression systems.
- Immunized mice with CD14-expressing cells.
- Fused spleen cells with myeloma cells to create hybridomas.
- Screened hybridomas for CD14-specific antibody production.
Main Results:
- Successfully generated 5 hybridoma clones secreting CD14-specific MAbs.
- The MAbs showed strong reactivity with monocytes, weak with neutrophils, and no reactivity with lymphocytes.
- The generated MAbs inhibited lipopolysaccharide (LPS) binding to CD14.
- The MAbs can be used for enumerating peripheral blood monocytes.
Conclusions:
- An alternative and effective antigen preparation method for MAb production against cell surface molecules was established.
- The novel CD14 MAbs are suitable for identifying monocytes and studying endotoxin interactions.
- This approach offers a valuable tool for immunological research and diagnostics.
Abstract:
CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.
