Measurement of antibody binding to intact bacteria using flow cytometric techniques

Laboratory based assays are used routinely to measure antibody binding to bacteria of their subunits and have become extremely important to the study of immune responses to these pathogens. We have developed a two-color fluorescence flow cytometric assay to measure the binding of bovine antibodies to intact Brucella abortus. Intact, irradiated B. abortus organisms, strain 19, were incubated with bovine plasma from B. abortus strain 19 vaccinated cattle. Bacteria with bound bovine immunoglobulin were labeled using an FITC conjugated anti-bovine immunoglobulin antiserum (green fluorescence), fixed to stabilize the antibody binding and to permeabilize the bacterial cell wall and then treated with propidium iodide (red fluorescence) to label the bacterial DNA. Dual labeled bacteria were analyzed with a flow cytometer using red fluorescence to identify bacteria from sample debris and green fluorescence to measure antibody binding. The advantages of such an assay are that intact bacteria can be used, labeling procedures are simple and readily automated and data represents the measurement of antibodies bound to a large number of individual bacteria which are used to determine the total antibody binding estimate. This assay was used to measure specific antibody levels using a single dilution of test plasma and was subject to less intertest variation than an enzyme linked immunosorbent assay.