Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules

Abstract

Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases.

Establishment of monoclonal antibodies to evaluate the cellular immunity in a hamster model of L infantum infection

“…Some studies have reported that anti‐mouse CD4 clone GK1.5 is able to stain 14% to 42% of total hamster´s splenocytes. Our results showed that 20%‐30% of alive splenocytes were CD4 + which match with the literature data 9,13,14 . Regarding intracellular cytokines, we chose three key cytokines that orchestrate the immune response in VL disease.…”

“…Based on previous studies, 7‐9 two mAbs anti‐CD4 and one mAbs anti‐CD8 were selected. Fixable viability stain 450 (FVS450, BD Biosciences) and commercially available rat anti‐mouse mAbs against TNF‐α, IFN‐γ and IL‐10 cytokines were also used (Table 1).…”

“…The possible reason for that is because of limited availability of immunological reagents and monoclonal antibodies (mob's) to study the immune system in this model. Hereupon, search for cross‐reactivity of available mAbs between different species has been suggested as a valuable strategy to obtain reagents for immunological studies in infectious disease 7‐9 . Previous studies have proposed the use of commercial anti‐mouse mAbs to phenotype blood, spleen and bone marrow cells in hamsters seeking out of the cross‐reactivity of these mAbs.…”

“…Previous studies have proposed the use of commercial anti‐mouse mAbs to phenotype blood, spleen and bone marrow cells in hamsters seeking out of the cross‐reactivity of these mAbs. Therefore, it was possible to stain CD4+ cells and the intracellular cytokine IFN‐γ 7‐9 …”

Improving immunological insights into the ferret model of human viral infectious disease

“…For example, prime‐boost immunisation using DNA and adenoviral‐based influenza vaccines provided effective protection in experimentally challenged ferrets, with protection correlating to the capacity of CD3+ T cells to express interferon gamma (IFN‐ γ) following in vitro stimulation on peripheral blood mononuclear cells (PBMCs) with HA peptide pools . Caution should be taken when using antibodies developed for other species in ferret experiments, as there may be subsets of cells displaying variable reactivity to the antibodies . In addition, currently available anti‐ferret B‐cell antibodies such as CD79α target intracellular epitopes, requiring fixation and permeabilisation.…”