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Development of Monoclonal Antibody Specific to Burkholderia pseudomallei for Diagnosis of Melioidosis
AuthorHandayani, Farida Dwi
Biochemistry and Molecular Biology
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Abstract Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative intracellular bacterium. This pathogen is a major contributor to community-acquired septicemia in Southeast Asia and Northern Australia, and notoriously difficult to diagnose. At present, a goal of our laboratory is to develop an immunoassay capable of accurately detecting shed or secreted bacterial antigens in patient body fluids. Here, we report on a group monoclonal antibodies (mAbs) that have been generated against B. pseudomallei for this purpose. mAbs 3C5 and 4C7 were found to react with the capsular polysaccharide (CPS) and lipopolysaccharide (LPS), respectively. This was confirmed by the inability of 3C5 to react with a B. pseudomallei CPS-deletion mutant, and its insensitivity to proteinase K treatment. mAb 4C7 was reactive with a 25-75 kD polysaccharide in a ladder pattern, a characteristic of LPS binding. mAb F5C3 is reactive with the B. pseudomallei flagellin protein monomer at 43 kD, as was confirmed by mass spectrometry of the reactive protein. We determined the specificity of mAbs 3C5, 4C7 and F5C3 with lysate from 12 various Burkholderia species and other common pathogenic bacteria. mAb 3C5 was reactive with B. mallei, but not B. thailandensis (both of these bacteria are closely related to B. pseudomallei) and mAb 4C7 was reactive with both B. mallei and B. thailandensis. mAb F5C3 was not reactive with B. mallei but was reactive with B. thailandensis although, at a slightly lower molecular weight (41 kD). All of the mAbs were tested for their ability to detect antigen in serum and urine samples from melioidosis patients. Antigen was not detected in serum, however, mAbs 3C5 and F5C3 were determined to be superior to mAb 4C7 in their ability to detect antigen in urine.