Detection of microbial antigens in clinical samples can lead to rapid diagnosis of an infection and administration of appropriate therapeutics. A major barrier in diagnostics development is determining which of the potentially hundreds or thousands of antigens produced by a microbe are actually present in patient samples in detectable amounts against a background of innumerable host proteins. In this report, we describe a strategy, termed in vivo microbial antigen discovery (InMAD), that we used to identify circulating bacterial antigens. This technique starts with “InMAD serum,” which is filtered serum that has been harvested from BALB/c mice infected with a bacterial pathogen. The InMAD serum, which is free of whole bacterial cells, is used to immunize syngeneic BALB/c mice. The resulting “InMAD immune serum” contains antibodies specific for the soluble microbial antigens present in sera from the infected mice. The InMAD immune serum is then used to probe blots of bacterial lysates or bacterial proteome arrays. Bacterial antigens that are reactive with the InMAD immune serum are precisely the antigens to target in an antigen immunoassay. By employing InMAD, we identified multiple circulating antigens that are secreted or shed during infection using Burkholderia pseudomallei and Francisella tularensis as model organisms. Potential diagnostic targets identified by the InMAD approach included bacterial proteins, capsular polysaccharide, and lipopolysaccharide. The InMAD technique makes no assumptions other than immunogenicity and has the potential to be a broad discovery platform to identify diagnostic targets from microbial pathogens.

The CDC Tier 1 select agent Francisella tularensis is a small, Gram-negative bacterium and the causative agent of tularemia, a potentially life-threatening infection endemic in the United States, Europe and Asia. Currently, there is no licensed vaccine or rapid point-of-care diagnostic test for tularemia. The purpose of this research was to develop monoclonal antibodies (mAbs) specific to the F. tularensis surface-expressed lipopolysaccharide (LPS) for a potential use in a rapid diagnostic test. Our initial antigen capture ELISA was developed using murine IgG3 mAb 1A4. Due to the low sensitivity of the initial assay, IgG subclass switching, which is known to have an effect on the functional affinity of a mAb, was exploited for the purpose of enhancing assay sensitivity. The ELISA developed using the IgG1 or IgG2b mAbs from the subclass-switch family of 1A4 IgG3 yielded improved assay sensitivity. However, surface plasmon resonance (SPR) demonstrated that the functional affinity was decreased as a result of subclass switching. Further investigation using direct ELISA revealed the potential self-association of 1A4 IgG3, which could explain the higher functional affinity and higher assay background seen with this mAb. Additionally, the higher assay background was found to negatively affect assay sensitivity. Thus, enhancement of the assay sensitivity by subclass switching is likely due to the decrease in assay background, simply by avoiding the self-association of IgG3.

The genus Burkholderia includes many bacteria that cause serious human infections. As is the case with other Gram-negative bacteria, Burkholderia species produce LPS, which is an abundant component of the bacterial cell surface. Burkholderia cepacia complex (Bcc) bacteria (which include at least 17 separate species) produce LPS structures that are quite different. In an attempt to determine the degree of LPS epitope variation among Bcc species, a mAb was produced, designated 5D8, specific for the LPS of B. cepacia. Western blot analysis determined that mAb 5D8 was able to produce the classic 'ladder pattern' when used to probe B. cepacia and Burkholderia anthina lysates, although 5D8 did not produce this pattern with the other seven Bcc species tested. mAb 5D8 reacted with varying intensity to most but not all of the additional B. cepacia and B. anthina strains tested. Therefore, there seems to be significant epitope variation among Bcc LPS both between and within species. Additionally, mAb 5D8 reacted with a proteinase-K-sensitive 22 kDa antigen in all Bcc strains and also in a strain of Burkholderia pseudomallei. INTRODUCTIONBacteria in the genus Burkholderia are Gram-negative bacilli that include species with the potential to cause lifethreatening human infections. People with cystic fibrosis (CF) are susceptible to infection by several Burkholderia species comprising the Burkholderia cepacia complex (Bcc) (LiPuma, 2005). 'Cepacia syndrome', which is characterized by necrotizing pneumonia and septicaemia resulting in rapid clinical deterioration, can develop in a small percentage of infected CF patients (Mahenthiralingam et al., 2005).Although much remains unknown about the pathogenic mechanisms of Burkholderia, these species produce a number of putative virulence determinants. One such determinant is LPS, which is present in abundance on the outer membrane of Gram-negative bacteria. Bcc LPS has potent endotoxic activity and elicits significantly higher levels of pro-inflammatory cytokines such as tumour necrosis factor, interleukin-6 and interleukin-8 than LPS from Pseudomonas aeruginosa, another common CF pathogen (Shaw et al., 1995;Vinion-Dubiel & Goldberg, 2003).The O antigen or O-specific polysaccharide, along with an oligosaccharide core and a lipid A molecule, are components of LPS (De Soyza et al., 2008). Bcc species produce many structurally distinct LPS O antigens (Burtnick et al., 2002;Vinion-Dubiel & Goldberg, 2003). In fact, 16 Oantigen serotypes have been identified among Bcc isolates recovered from CF patients (Nakamura et al., 1986;Soldatkina et al., 1989;Vinion-Dubiel & Goldberg, 2003). These serotypes do not correlate with Bcc species (Kenna et al., 2003;Rabkin et al., 1989). As a consequence, Oantigen serotype cannot be used to determine the specific Bcc species causing an infection. The O-antigen biosynthetic genes of Burkholderia stabilis (a member of the Bcc) have been mapped based on homology to other polysaccharide biosynthetic genes (Vinion-Dubiel & Goldberg, 2003). One gene, wbiI, wa...