Fig. 2.

Sera of BNP dams contain alloreactive antibodies that bind surface antigens on bovine leukocytes. (A) Bovine leukocytes were isolated from whole blood of a 17 days old calf and analyzed by flow cytometry. An exemplary FACS analysis is shown: the upper left panel shows the gating strategy. According to forward and sideward scatter lymphocytes (L¢), monocytes (M¢) and granulocytes (G¢) are differentiated. This is confirmed by additional staining: the upper middle panel shows the expression of CD3 for the lymphocyte gate, the upper right panel shows the expression of CD14 for the monocyte gate. Dotted lines represent isotype controls. The lower panel depict fluorescence intensity histograms obtained with serum from a BNP dam (bold line) or a non-immunized control (dotted line) for the respective leukocyte populations. Numbers indicate the percentage of the population that does not overlap with the control. (B) The same serum panel as in Fig. 1 was tested by flow cytometry. Results are shown for PBMCs from one representative leukocyte donor, a 2-week-old calf. Black dots represent the MFI within the lymphocyte gate for individual sera, black bars indicate the median value for each group. P-Values were calculated by simple Student's t-test. (C) A panel of sera from seven BNP dams (filled columns) and three non-immunized controls (empty columns) was tested for alloreactivity against PHA blasts from three different leukocyte donors. Donor A and B were adult cows, Donor C was a young bull of 7 months age. Sera are always shown in the same order. (D) The same serum panel as in Fig. 1 was tested by Western blot. Whole cell lysates of PHA blasts from a 1-year-old heifer were separated by non-denaturing SDS-PAGE and blotted onto nitrocellulose membranes. For each group eight sera were investigated and the membrane strips are grouped accordingly.