Abstract
BackgroundThe process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49–96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed.ResultsEleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies.ConclusionThe identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.
Highlights
The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories
Anti‐human cytokine monoclonal antibodies (mAb) cross‐reactivity against canine cytokines The analysis of the frequencies of lymphocytes expressing intracytoplasmic cytokines were determined by the strategy of conventional analysis as described using TNF-α as an example (Fig. 1)
Higher production of some cytokines such as IL-8, IL-12, IL-17A and TNF-α was observed in the PMA + LPS stimulated cultures as compared with control cultures
Summary
The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. Cytokines are soluble proteins secreted by different cell subtypes including antigen-presenting cells (APC), endothelial and epithelial cells, bone marrow stromal cells, fibroblasts, keratinocytes, platelets and both lymphoid and non-lymphoid cells. They are involved in a wide range of interactions such as the development of cellular and humoral immune responses, induction of inflammatory responses, regulation of hematopoiesis, control of proliferation/differentiation and cell migration [1]. There is great demand for reactive antibodies against canine molecules, especially anti-cytokines
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