Cross-reactive anti-human monoclonal antibodies as a tool for B-cell identification in dogs and pigs.

We have characterized a panel of commercially available anti-human monoclonal antibodies (mAbs) suitable for B-cell identification in pigs and dogs. The specificities of the mAbs were against CD20, CD21, CD22, and CD86. In addition to HM57, originally raised against human CD79alpha the broad cross-reactivity of which was documented more than 10 years ago, we recommend here a panel of several other mAbs as a useful tool for immunophenotyping and multicolor flow cytometry of canine and porcine B-lymphocytes. All six investigated antibodies did bind weakly to either canine or porcine lymphocytes (or both), but considerable weaker than for the human control cells. Four of them did bind to canine or porcine spleen section in immunohistochemistry. Monoclonal antibody against CD22 (clone RFB-4) was the only antibody in the tested panel the cross-reactivity of which was confirmed by Western blot. The advantages and limits of cross-reactive mAbs in studies on animal B-cells are discussed.

Commercially available rabbit anti-human polyclonal antisera as a useful tool for immune system studies in veterinary species.

We have used selected rabbit anti-human polyclonal antibodies as an example of useful and easily available tools for studies on immune system structure and development in important veterinary species, many of which also represent animal models in biomedicine. The cocktail of anti-human Igkappa-FITC/anti-Iglambda-RPE F(ab')(2) fragments was used for two-colour and, in combination with the cross-reactive anti-CD79alpha monoclonal antibody HM-57, for three-colour flow cytometry of canine, feline, bovine and porcine peripheral B-cells. A possible application of such immunoreagents in studies on primary B-cell differentiation has been suggested in pigs; the same approach can be used in other species of interest. Rabbit anti-human lactoferrin-FITC F(ab')(2) fragment was used for visualizing neutrophils in dogs, pigs and cattle and an application for two-colour immunophenotyping of canine granulocyte subsets has been designed. Affinity isolated rabbit anti-human CD3 and anti-human TdT have been shown to represent a ready-to-use tool for in situ studies on primary T-lymphopoiesis in pigs with possible extensions both to the B-lineage development in pigs and other animal models. Altogether, our study show that carefully selected polyclonal antibodies available on the market may possess broad cross-reactivity with important applications in veterinary research.

Early postnatal development of the immune system in piglets: the redistribution of T lymphocyte subsets.

In this study, we have characterised postnatal changes in T lymphocyte subsets, especially gammadelta T lymphocytes, in blood, spleen and lymph nodes. Detection was carried out using two-colour flow cytometry and three-colour immunohistochemistry. During ontogeny, there was a significant increase in the total percentage of gammadelta T cells in the spleen and blood. In the lymph nodes, there were no age-dependent changes in the total percentage of gammadelta T cells, but the percentage of the gammadeltaTCR+CD8+ subpopulation significantly increased. The tissue distribution of gammadeltaTCR+CD8+ and gammadeltaTCR+CD8- cells in the lymph nodes is random and not collocated with a particular area of the organ. Furthermore, postnatal development was characterised by an increasing frequency of CD8+CD3+CD4-gammadeltaTCR-, which was compensated by a decreasing proportion of CD4+ lymphocytes. Double positive CD4+CD8+ lymphocytes were rare during the first month of life and a significant age-dependent increase of these cells was found in all the compartments monitored.