Recombinant Truncated Flagellin of Burkholderia pseudomallei as a Molecular Probe for Diagnosis of Melioidosis

Abstract

Current serological tests for melioidosis, using impure or uncharacterized cell antigens from Burkholderia pseudomallei, have problems in detection sensitivity and specificity. Therefore, we designed and expressed the recombinant flagellin (truncated at both the N-and C-terminal ends), and used the antigen to develop an indirect enzyme-linked immunosorbent assay (ELISA) to diagnose melioidosis. Comparison of the immunoreactivities of the full-length and truncated flagellins reveals that the truncated flagellin performed much better in detection specificity and sensitivity. Only the full-length flagellin was recognized by other bacterial causing septicemia and gave a false-positive result in Western analysis, indicating that the cross-reactive epitopes were located on the more highly conserved N-and C-terminal regions of flagellin. The indirect ELISA using recombinant truncated flagellin as the antigen achieved 93.8% sensitivity and 96.3% specificity and offered a more efficient serodiagnosis of melioidosis.

CpG-Modified Plasmid DNA Encoding Flagellin Improves Immunogenicity and Provides Protection againstBurkholderia pseudomalleiInfection in BALB/c Mice

“…Briefly, the 96-well microplates were coated with flagellin (0.5 g/ml) overnight at 4°C. The flagellin preparation was performed according to a procedure described in a previous report (7). The contaminant LPS was removed from the flagellin preparation by passage through a polymyxin B column according to the manufacturer's instructions (Detoxi-Gel; Pierce Chemical Co., Rockford, IL).…”

“…The oligodeoxynucleotides (ODNs) as synthesized by MdBio Inc. (Taipei, Taiwan) are made up of DNA consisting of an unmethylated CpG motif (5Ј-TCT CCC AGC GTG CGC CAT-3Ј) and based upon the work of Elkins et al (13). The mammalian expression vector pcDNA3/fliC encoding the B. pseudomallei flagellin was created from pGEX4T-2/fliC using appropriate restriction sites (7). This unmethylated ODN (containing two CpG motifs) was added to the plasmid sequence using BamHI linkers on the oligonucleotide sequence of the ODN (Fig.…”

“…The amplified products were analyzed on a 1% agarose gel. The cell culture was also used to prepare total cell extracts, and then immunoblotting was performed to analyze the specific gene expression (7).…”

Immunostimulatory Flagellin from Burkholderia pseudomallei Effects on an Increase in the Intracellular Calcium Concentration and Up‐Regulation of TNF‐α by Mononuclear Cells

“…This was repeated until the concentration of LPS was less than 1 pg/ml as determined by the Limulus Amebocyte Lysate assay (BioWhittaker, Inc., Walkersville, Md., U.S.A.). Polyclonal anti-flagellin antibodies were obtained as described previously (4). These antibodies were purified using a protein ASepharose CL-4B column (Sigma) and also had any LPS contamination removed by a polymyxin B column.…”

“…Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by centrifugation over a FicollHypaque gradient (Sigma Chemical Co., St. Louis, Mo., U.S.A.) for 10 min at 700ϫg. The red blood cells were removed by washing with 0.83% NH 4 Cl. The PBMCs were recovered and washed twice with RPMI 1640 (GIBCO BRL, Rockville, Md., U.S.A.) containing 10% fetal calf serum (FCS).…”