Generation and validation of novel anti-bovine CD163 monoclonal antibodies ABM-1A9 and ABM-2D6.

ABSTRACT

The scavenger receptor CD163 is widely used as a cell signature for alternatively active "M2" macrophages in mammals. In this study, we generated two monoclonal antibodies, ABM-1A9 and ABM-2D6, against the extracellular region of bovine CD163. Conventional Western blotting using the antibodies yielded immunoreactive bands of bovine CD163 at 130 and 150 kDa in non-reduced and reduced spleen lysates, respectively. The minimum limit of detectable concentration of both antibodies was relatively lower (5.0 ng/mL) than that of the anti-human CD163 monoclonal antibody AM-3 K (>1.0 µg/mL), which has been used previously for the detection of bovine CD163. An immunohistochemical study using formalin-fixed paraffin-embedded sections revealed that ABM-1A9 and ABM-2D6 clearly stained some Iba1+ macrophages in the lymph nodes of cattle with mastitis. Moreover, the CD163-stained macrophages were frequently observed engulfing leukocytes. ELISA using ABM-2D6 distinguished levels of circulating soluble CD163 in healthy cattle (less than 16.9 pmol/mL) and cattle with mastitis (more than 33.7 pmol/mL). These new monoclonal antibodies can be used in the diagnosis and evaluation of inflammatory disease prognosis in cattle with immunohistological analyses and blood test applications.