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Monoclonal antibodies identifying leukocyte antigens are highly important tools for studying equine leukocyte biology. The rational of the present study was to provide domestic and wild equine immunology with new antibodies to leukocyte markers filling gaps of available reagents and establish a leukocyte tool-box for some members of family Equidae. To achieve the target, a large panel of mostly commercially available anti-leukocyte mAbs was analyzed for reactivity with equid leukocytes and cell lines. Analysis of 534 mAbs demonstrated for 31 mAbs a clear positive reactivity, for 2 mAbs a weak positive reactivity; for 23 mAbs a positive "+"; but possibly valuable staining; for 15 mAbs an alternate expression pattern from that expected from human immunology; for 6 mAbs a questionable staining; for 45 mAbs a weak-positive reactivity and alternate expression pattern, and 356 mAbs remained negative. In 53 cases, more appropriate target cells, such as thymocytes or bone marrow stem cells, were not available for screening. Positive mAbs were directed against CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD34, CD44, CD45, CD49d, CD68, CD83, CD91, CD163, CD172a, CD206, CD283, MHCI, MHCII, and canine B cell molecule. 22 mAbs spanning 16 different CD antigens were analyzed with Somali wild ass (Equus africanus somalicus), Grevys zebra (Equus grevyi), and Hartmanns mountain zebra (Equus zebra hartmannae) leukocytes using one colour flow cytometry. Most but not all mAbs cross reacted with the three species. Cross-reactive mAbs analyzed at this part of the study may be considered the basis to establish an immune tool-box for these wild equids but require further analysis. In addition, a broad panel of mAbs predominantly reactive with equine PBMC were applied on equine cell lines (EqT8888 and eCAS). A limited number of these mAbs reacted positively with the equine cell lines. Based on the obtained data, eCAS may represent a very early stage of a myeloid cell line and EqT8888 may be a B cell lymphoma. Analysis of CD28 was performed for some members of the order Perissodactyla, including three members of family Equidae: Somali wild ass, Hartmanns mountain zebra, and Grevys zebra. In addition, a member of family Rhinocerotidae, the Greater one-horned rhinoceros (Rhinoceros unicornis) was included beside the Asian elephant (Elephas maximus) representing order Proboscidae, family Elephantidae and members of order Artiodactyla, family Bovidae, such as European bison (Bison bonasus) and African Buffalo (Syncerus caffer) and one member of family Giraffidae, Nubian Giraffe (Giraffa camelopardalis). Comparison of CD28 aa sequence revealed a high degree of interspecies conservation with aa identity ranging from 68-99%. Animal sequences were more closely related between each other than to humans and mice represented the out-group. 18 aa mismatches between equids and human were detected in the extracellular domain and could explain the inability of six human CD28 specific clones to stain horse leukocytes. Anti-human polyclonals were used as another tool to detect important equine CD antigens like CD28 and CD25 to which none of the submitted mAbs were positive with horse leukocytes. The anti-human CD28 polyclonal Ab detected a protein likely to be EqCD28 on a small population of lymphocytes in flow cytometry only, but precipitated it as two proteins of 57 and 46 kDa from surface biotinylated horse lymphocytes. Cloning and expression of EqCD28 in insect cells also resulted in two proteins of 57 and 46 kDa. Equine CD25 expression was detected using a goat anti-human IL-2R polyclonal antibody in flow cytometry. In addition, equine CD25 was immunoprecipitated using this Ab as a molecule of approximately 55 kDa molecular weight which is analogous to human CD25. Although just a few mAbs were positive on equine cells (31/534), the approach may be considered useful, especially as their specificity has been resolved in this study. In addition, this study supports the use of highly purified anti-human polyclonal antibodies as an alternative approach in the analysis of equine leukocyte if their cross-reactivity can be confirmed and background signals eliminated.