Abstract
Antigens produced from Brucella rough lipopolysaccharide (rLPS) antigens have the potential to aid in the serodiagnosis not only of infection due to rough species and strains of Brucella but also infection due to smooth strains. In the latter case this would assist in resolving samples where a false positive result against smooth antigens (i.e. those containing O polysaccharide) may be suspected. A ‘Rough Antigen’ extract prepared from B. abortus RB51 using phenol/chloroform/petroleum ether (PCP) has been evaluated for the serodiagnosis of porcine (B. suis) and canine brucellosis (B.canis) and found to be highly effective. Analysis of the PCP fraction revealed it to be a protein free mixture of rLPS (~60%) and outer membrane phospholipids (~40%). We investigated if further purification of the rLPS may yield a diagnostically superior antigen and produced an antigen that was higher in rLPS purity (>95%). However, this antigen was diagnostically inferior. Furthermore, the depleted material (~5% rLPS) remained diagnostically similar to the original PCP extract. Fragmentation Mass Spectrometry of the depleted antigen suggested the predominance of phospholipids (including phosphatidyl choline) and ornithine lipids. Thin Layer Chromatography (TLC) of the depleted antigen confirmed this predominance and elution of the TLC fractions enabled the specific components to be tested by ELISA. This evaluation confirmed that the major antibody binding material in the extract was rLPS rather than any of the co-extractants. This finding raised the possibility that the presence of surfactants such as phospholipids is enabling and enhancing the diagnostic efficacy of the rLPS in the ELISA. To investigate this we evaluated a range of surfactants by using them to dilute the purified rLPS. The ELISA result from these dilutions showed no increase in the response to a positive serum in the case of dilution with octyl glucoside and CHAPS, but an increase of tenfold or more in the titre of the response after dilution with phospholipids or dodecylmaltoside. We believe that this observation is related to the critical micellar concentration of these compounds and demonstrates that for some antigens, the inclusion of non-antibody binding components can enhance diagnostic performance.
