Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and immunohistochemistry

Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules from primary equine PBMC or the equine cell line T8888 were biotinylated prior to precipitation to determine the molecular weight of the corresponding molecules in a western blot using streptavidin-AP. 21 out of 24 mAbs precipitated the molecules with a MW corresponding to its human orthologue. Positive mAbs were directed against CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD44, CD83, CD91, CD172a, MHCI and MHCII. Three mAbs directed against CD49d, CD163, and CD206 which were unambiguously identified earlier by flow cytometry failed to immunoprecipitate the corresponding CD molecule. MAbs detecting CD molecules which are expressed internally like CD68 and mAbs of IgM class could not be included into this approach.