Bacterial fractionation flow chart.

<p>Bacteria were sonicated and fractionated into subcellular components: cell wall, cytosol, and membrane. The membrane antigen preparation was not further pursued. Cell wall associated proteins were extracted with SDS and both the cytosol and cell wall fractions were then extracted with TX-114 to remove non-specific hyporesponsive antigens, mostly LAM, LM, and PIM's. Residual detergent was removed by affinity chromatography. Antigens were diluted to prescribed concentrations, vialed, labeled, and autoclaved.