# Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9
## 1 Introduction  
S100A9 is a member of the calcium-binding S100 protein family, which participates in cytoskeleton rearrangement and arachidonic acid metabolism [1]. As S100A9 is secreted at high concentrations from immigrated and activated phagocytic cells during inflammation [2], it plays a critical role in modulating the inflammatory response by stimulating leukocyte recruitment or inducing cytokine secretion [1]. S100A9 mediates the inflammatory process in myeloid cells and is involved throughout the initial development of cancer cells and metastatic disease progression [3, 4]. Moreover, S100A9 is involved in the formation of amyloid aggregates, integrating the amyloid-neuroinflammatory cascade in [PERSON]'s disease, and enhancing the neurodegenerative effects of Parkinson's disease [5, 6, 7, 8]. Elevated levels of S100A9 have also been noted in chronic bronchitis, cystic fibrosis, systemic lupus erythematosusous, rheumatoid arthritis, colitis ulcerosa, [PERSON]'s disease, inflammatory bowel disease, colorectal cancer, multiple sclerosis, and human immunodeficiency virus infection [9, 10, 11, 12]. Detection strategies are needed if S100A9 is to be considered a biomarker of these diseases.  
Antibody assays are the method of choice for disease diagnosis and treatment because they strongly bind to the target antigen, even in complex blood or tissue samples. Antibodies are classified as polyclonal or monoclonal, and hybridoma technology is a traditional and robust method for generating antibodies. Antibody detection tests are cost effective and are neither labor intensive nor technically demanding compared withthe more expensive and complex phage display technology. Polyclonal antibodies are purified from the serum of an immunized animal, and monoclonal antibodies are secreted by immortalized B cells from the spleen of an immunized animal. In order to generate a unique monoclonal antibody of the desired specificity, the splenic B cells are fused with myeloma cells, resulting in immortal hybridomas [13]. Although polyclonal antibodies can be generated more rapidly, at less expense, and with less technical skill than is required to generate hybridomas and, subsequently, monoclonal antibodies, the principal advantages of monoclonal antibodies are their homogeneity and consistency, whereas polyclonal antibodies are heterogeneous. Specifically, as a monoclonal antibody recognizes a single epitope of an antigen, its affinity and selectivity to the target antigen are greater than those of a polyclonal antibody. Another advantage of monoclonal antibodies is that once the desired hybridoma has been generated, it becomes a constant and renewable resource. In contrast, polyclonal antibodies generated to the same antigen using multiple animals will differ between individuals, and their avidity may change as they are harvested over time. Therefore, the concentration and purity levels of specific antibodies are higher in monoclonal than in polyclonal antibodies. A few commercially available purified anti-S100A9 monoclonal antibodies have been developed. However, to the best of our knowledge, no study has been published on the production and sequencing of monoclonal anti-S100A9 antibodies.  
We aimed to generate and sequence a monoclonal antibody against S100A9. The sequence information can be used for the development of recombinant antibodies or antibody fragments against S100A9. Additionally, the detailed methods for antigen production, antibody generation, and sequencing will support other efforts to produce monoclonal anti-S100A9 antibodies for use as a biological reagent to bind S100A9 in diagnostic and therapeutic approaches.