Titres of melioidosis haemagglutinating antibodies of 1/40 or more were found in 18 of 905 British, Australian, and New Zealand soldiers serving in West Malaysia. Previous mild unsuspected melioidosis seemed to be responsible for these positive titres, which were more common in men exposed to surface water at work and during recreation. This accords with the current view that soil and surface water is the normal habitat of Pseudomonas pseudomallei, the causal organism. Pyrexia of unknown origin after arriving in Malaysia was significantly more common in men with titres of 1/40 or more than in the remainder. It is suggested that mild melioidosis may present as pyrexia of unknown origin. Pyrexias of unknown origin should be investigated vigorously in patients who are in or who have visited endemic areas.
A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.
A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30 degrees C until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30 degrees C for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.