Evaluation of cross-reactivity of monoclonal antibodies against cytokines and cellular molecules of bovine and human origin to identify immunological markers in buffaloes (Bubalus bubalis).

Research related to the investigation of the activation and regulation of the immune system of domestic buffaloes is still very limited. The main factor linked to this limitation is the scanty availability of commercial monoclonal antibodies (mAb) against buffalo immune markers that allow the use of multiparametric techniques, such as flow cytometry, to study the modulation of the immune system of these animals. The evaluation of the cross-reactivity of commercially available mAbs against other species for cytokines and components of the immune system of buffaloes, is the fastest and least expensive way to expand the tools to assess immune response in those animals. Therefore, sixteen commercial mAbs anti-cell markers (CD4, CD8, CD14, CD21, CD25, CD28, CD45RB, CD45RO, WC1, and CD80) and anti-cytokines or other effector molecules of the immune system (TNF-α, IFN-γ, IL-4, IL-17A, granzyme B and perforin) that recognize molecules from bovine and human species were evaluated in whole blood samples from buffaloes and cattle. Two experiments were performed: Experiment 1, with six buffaloes, evaluated the specificity of mAb binding, and experiment 2, with twelve buffaloes and twelve cattle, evaluated the cross-reactivity of the mAbs. Samples were processed and analyzed by flow cytometry. Of the mAbs analyzed 15 clones were validated for flow cytometry of buffalo cells; only the CD4 clone did not show cross-reactivity to buffaloes amplifying the spectrum analyses of immune response in this animal species. This validated panel enables standardized flow cytometric immunophenotyping in buffaloes for research on vaccination, infectious disease, and immune regulation.