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Development of Immunoassays for Burkholderia pseudomallei Typical and Atypical Lipopolysaccharide Strain Typing

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ABSTRACT

Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection endemic to many tropical regions. Lipopolysaccharide (LPS) is recognized as an important virulence factor used by B. pseudomallei. Isolates of B. pseudomallei have been shown to express one of four different types of LPS (typical LPS, atypical LPS types B and B2, and rough LPS) and in vitro studies have demonstrated that LPS types may impact disease severity. The association between LPS types and clinical manifestations, however, is still unknown, in part because an effective method for LPS type identification is not available. Thus, we developed antigen capture immunoassays capable of distinguishing between the LPS types. Mice were injected with B or B2 LPS for atypical LPS–specific monoclonal antibody (mAb) isolation; only two mAbs (3A2 and 5B4) were isolated from mice immunized with B2 LPS. Immunoblot analysis and surface plasmon resonance demonstrated that 3A2 and 5B4 are reactive with both B2 and B LPS where 3A2 was shown to possess higher affinity. Assays were then developed using capsular polysaccharide–specific mAb 4C4 for bacterial capture and 4C7 (previously shown to bind typical LPS) or 3A2 mAbs for typical or atypical LPS strain detection, respectively. The evaluations performed with 197 strains of Burkholderia and non-Burkholderia species showed that the assays are reactive to B. pseudomallei and Burkholderia mallei strains and have an accuracy of 98.8% (zero false positives and two false negatives) for LPS typing. The results suggest that the assays are effective and applicable for B. pseudomallei LPS typing.

No MeSH data available.


Related in: MedlinePlus

Surface plasmon resonance (SPR) analysis of binding affinity between monoclonal antibodies (mAbs) 3A2 and 5B4 to immobilized type B lipopolysaccharide (LPS). Biotinylated LPS type B was immobilized on the surface of streptavidin (SA)-coated sensor chip with the final response units of 306.2. The sensorgrams (left panel) were obtained by injecting the mAbs 3A2 (10–333 nM, Panel A) and 5B4 (67–6,667 nM, Panel B) over the chip surface for 180 seconds followed by passive dissociation for 300 seconds. Right panel presents steady-state affinity model fitting of each mAb.
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fig2: Surface plasmon resonance (SPR) analysis of binding affinity between monoclonal antibodies (mAbs) 3A2 and 5B4 to immobilized type B lipopolysaccharide (LPS). Biotinylated LPS type B was immobilized on the surface of streptavidin (SA)-coated sensor chip with the final response units of 306.2. The sensorgrams (left panel) were obtained by injecting the mAbs 3A2 (10–333 nM, Panel A) and 5B4 (67–6,667 nM, Panel B) over the chip surface for 180 seconds followed by passive dissociation for 300 seconds. Right panel presents steady-state affinity model fitting of each mAb.

Mentions: SPR was used to study binding affinity of mAbs 3A2 and 5B4 for LPS. Each mAb was analyzed at several concentrations over the surface of a type B LPS–coated sensor chip (Figure 2Figure 2.


Development of Immunoassays for Burkholderia pseudomallei Typical and Atypical Lipopolysaccharide Strain Typing
Surface plasmon resonance (SPR) analysis of binding affinity between monoclonal antibodies (mAbs) 3A2 and 5B4 to immobilized type B lipopolysaccharide (LPS). Biotinylated LPS type B was immobilized on the surface of streptavidin (SA)-coated sensor chip with the final response units of 306.2. The sensorgrams (left panel) were obtained by injecting the mAbs 3A2 (10–333 nM, Panel A) and 5B4 (67–6,667 nM, Panel B) over the chip surface for 180 seconds followed by passive dissociation for 300 seconds. Right panel presents steady-state affinity model fitting of each mAb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5303037&req=5

fig2: Surface plasmon resonance (SPR) analysis of binding affinity between monoclonal antibodies (mAbs) 3A2 and 5B4 to immobilized type B lipopolysaccharide (LPS). Biotinylated LPS type B was immobilized on the surface of streptavidin (SA)-coated sensor chip with the final response units of 306.2. The sensorgrams (left panel) were obtained by injecting the mAbs 3A2 (10–333 nM, Panel A) and 5B4 (67–6,667 nM, Panel B) over the chip surface for 180 seconds followed by passive dissociation for 300 seconds. Right panel presents steady-state affinity model fitting of each mAb.
Mentions: SPR was used to study binding affinity of mAbs 3A2 and 5B4 for LPS. Each mAb was analyzed at several concentrations over the surface of a type B LPS–coated sensor chip (Figure 2Figure 2.

View Article: PubMed Central - PubMed

ABSTRACT

Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection endemic to many tropical regions. Lipopolysaccharide (LPS) is recognized as an important virulence factor used by B. pseudomallei. Isolates of B. pseudomallei have been shown to express one of four different types of LPS (typical LPS, atypical LPS types B and B2, and rough LPS) and in vitro studies have demonstrated that LPS types may impact disease severity. The association between LPS types and clinical manifestations, however, is still unknown, in part because an effective method for LPS type identification is not available. Thus, we developed antigen capture immunoassays capable of distinguishing between the LPS types. Mice were injected with B or B2 LPS for atypical LPS–specific monoclonal antibody (mAb) isolation; only two mAbs (3A2 and 5B4) were isolated from mice immunized with B2 LPS. Immunoblot analysis and surface plasmon resonance demonstrated that 3A2 and 5B4 are reactive with both B2 and B LPS where 3A2 was shown to possess higher affinity. Assays were then developed using capsular polysaccharide–specific mAb 4C4 for bacterial capture and 4C7 (previously shown to bind typical LPS) or 3A2 mAbs for typical or atypical LPS strain detection, respectively. The evaluations performed with 197 strains of Burkholderia and non-Burkholderia species showed that the assays are reactive to B. pseudomallei and Burkholderia mallei strains and have an accuracy of 98.8% (zero false positives and two false negatives) for LPS typing. The results suggest that the assays are effective and applicable for B. pseudomallei LPS typing.

No MeSH data available.


Related in: MedlinePlus