![]() Cells were incubated for 15 min at room temperature in the dark and then centrifuged as described above. Secondary antibodies were diluted in PBS containing 0.02% NaN 3 and 1% inactivated FBS. Supernatant was decanted and cells were labeled with 100 μL of a cocktail containing 4 secondary isotype-specific antibodies conjugated to fluorochromes (listed in Table 1). Cells were incubated for 15 min at room temperature and centrifuged (400 × g at room temperature for 2 min). Monoclonal primary antibody diluted in PBS containing 0.02% NaN 3 and 1% inactivated FBS was added to individual wells in 50 μL aliquots. Primary and secondary antibodies used for flow cytometric-staining are listed in Table 1. Expression of Activation Molecules on B Cells Isolated from Lymph Nodes The change in optical density readings was calculated by subtracting the mean optical density readings for wells receiving coating buffer or PBS alone from the mean optical density readings for antigen-coated wells receiving the same test sample. The reaction was stopped by addition of sulfuric acid (0.18 M) and absorbances (450 nm) of individual wells measured (ELISA plate reader, Molecular Devices, Menlo Park, CA). Wells were then washed with PBST and incubated for 4.5 min at room temperature with substrate (3,3′5,5′-tetramethylbenzidine Kirkegaard and Perry Laboratories). After incubating for 20 h at 4☌ with test samples, wells were washed with PBST and incubated for 1 h at 37☌ with horseradish peroxidase-conjugated antibovine IgG heavy and light chains (Kirkegaard and Perry Laboratories) in PBS plus 0.1% gelatin. Optimal dilutions of test sera were determined by evaluation of the reactivity of 2-fold serial dilutions ranging from 1:6 to 1:800. Test and control sera were diluted in PBS containing 0.1% gelatin. After incubation for 1 h at 37☌ in the blocking solution, wells were washed in PBST and test sera were added to wells (100 μL/well). Plates were washed 3× with PBS with 0.05% Tween 20 ( PBST 200 μL/well) and blocked with a commercial milk diluent/blocking solution (200 μL/well Kirkegaard and Perry Laboratories, Gaithersburg, MD). Plates, including control wells containing coating buffer or PBS alone, were incubated for 15 h at 4☌. Microtiter plates (96-well Immulon II, Dynatech) were coated with OVA or WCS-PK (100 μL/well). The concentration of WCS-PK used in the ELISA was 40 μg/mL diluted in carbonate/bicarbonate coating buffer (pH 9.6). ![]() The concentration of OVA used in the ELISA was 1.56 μg/mL diluted in PBS. The relative amounts of Ig to OVA and WCS-PK in serum and in culture supernatants were determined by a capture ELISA. Mycobacterium bovis strain bacillus Calmette-Guerin.Additional studies are necessary to determine whether maternal immunologic experience transferred via colostral immunoglobulin inhibits production of mycobacteria-specific immunoglobulin production in the calf. In conclusion, calves generated B cell responses to ovalbumin and BCG after vaccination. Interestingly, IgM + cells isolated from the superficial cervical lymph node draining the vaccination site, but not from the opposing cervical lymph node, responded to antigen stimulation in vitro. An increased expression of major histocompatibility class II on CD5 +IgM + cells after stimulation was the only exception. Expression of activation molecules on ovalbumin- and purified protein derivative-stimulated CD5 +IgM + cells was similar to expression on the larger IgM + cell population. Stimulation of the same population with purified-protein derivative increased CD25 and CD80 expression on IgM + cells. Lymph node cell populations stimulated with ovalbumin had decreased CD5, CD21, and CD40 expression and increased B-B2, CD25, and CD80 expression on IgM + cells. In the second experiment, 6 calves were vaccinated with ovalbumin at 3 and 5 wk of age in addition to BCG at 3 wk of age. Vaccination with BCG did not elicit a measurable antibody response. ![]() Vaccination of 3-wk-old calves with ovalbumin elicited antigen-specific IgG 1 and IgG 2 anti-body responses that were amplified by secondary vaccination. Ovalbumin-specific IgG 1 and IgG 2 were not detected before vaccination. Mycobacterium bovis lipoarabinomannan-reactive IgG 1 and IgG 2 were detected in calf sera prior to vaccination, indicative of colostral transfer of maternal Ig cross-specific to BCG. ![]() Three of the 6 calves also were vaccinated with Mycobacterium bovis, strain bacillus Calmette-Guerin (BCG) at 3 wk of age. In the first of 2 experiments, 6 calves were vaccinated with ovalbumin at 3 and 5 wk of age. The objective of this research was to evaluate the effects of early vaccination on the phenotype (i.e., activation marker expression) and functional capacity of B cell populations in neonatal calves. ![]()
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