Immunochemical and partial chemical characterization of fractions of membrane-bound smooth lipopolysaccharide-protein complex from Brucella abortus

Smooth lipopolysaccharide (sLPS) of Brucella abortus was prepared and fractionated by a modification of the procedures of Moreno et al. (J. Bac. 138:361–369, 1979). Washed B. abortus cells were disrupted by 21 freeze-quick thaw cycles with ultrasonication to separate the non-membrane-bound material. Ultrasonicated bacteria were used for preparation of membrane-bound sLPS ( f5, the main crude sLPS fraction described by Moreno et al.). Phenol extraction was repeated 3 times and then washed with H₂O 10 times to remove most of the chromogen, polysaccharides and nucleic acids, eliminating the need for enzyme treatment as described previously. The membrane-bound sLPS was fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation, these fractions required only 80 ng for positive ELISA, about 0.2 ng for positive Limulus lysate tests, and reacted well with precipitating antibodies in the serum from a strain 2308 infected cow. They had marked differences in precipitin curves and chemical composition. The protein content varied from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which implies that the proteins associated with LPS may also play important roles in the complex for the immunochemical interactions and the heterogeneity of B. abortus lipopolysaccharide protein complex. As compared with previous reports, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, was obtained. Group f5A, which had a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19 and 2308). The amount of other subfractions obtained varied with batches or strains of B. abortus. These results provide a new profile of the immunochemical reactivities and the heterogeneity on B. abortus smooth membrane-bound endotoxins.Abbreviations LPS lipopolysaccharides - sLPS smooth lipopolysaccharides - cLPS crude lipopolysaccharides - AH acid hapten - KDO 3-deoxyoctulosonic acid - ELISA enzyme-linked immunosorbent assay - f5 the main crude sLPS described by Mooreno et al. [2] - LAL limulus amoebocyte lysate test - HexN Hexosamine - PS phenol sulfuric method - O orcinol method