Antibodies against bacterial membrane proteins

Prior studies have shown that antiserum raised to the Escherichia coli J5 v accine binds to three bacterial outer membrane proteins (OMPs) that are pre sent on serum-exposed smooth Enterobacteriaceae and in LPS-containing fragm ents that are released by bacteria incubated in human serum. The present st udies were performed to further analyze release of the three OMPs into huma n serum. Normal human serum was incubated with E. coli O18K(+) bacteria and then filtered to remove intact bacteria. Lipopolysaccharide (LPS) was affi nity-purified from sterile filtrates using murine monoclonal IgG directed a gainst the O-polysaccharide chain of E. coli O18 LPS (monoclonal anti-O IgG ). Components of sterile filtrates were also separated by ultracentrifugati on on sequential potassium bromide and cesium chloride gradients. Samples w ere analyzed by immunoblotting, using anti-J5 IgG and polyclonal anti-O IgG as primary antibodies. Three OMPs, of MWs 5-9, 18, and 35 kDa were detecte d in samples that were affinity-purified using monoclonal anti-O IgG, indic ating that complexes containing at least these three OMPs and LPS (OMP/LPS complexes) are released into human serum. The 18 kDa OMP was detected in lo w density fractions of the density gradients, indicating that this OMP is a lso released separately from the OMP/LPS complexes in a form that floats at the same density as serum lipoproteins.