Kinetics for clearance of CPS from serum.

<p>Mice were intravenously injected with 100 μg, 20 μg, or 4 μg of CPS. Blood samples were collected at the designated time points, and CPS concentrations in serum samples were determined using quantitative sandwich ELISA. The data were best described by a two-parameter monophasic exponential decay model (<i>y = ae</i>^<i>-bx</i><i>)</i>, where <i>a</i> is the <i>Y</i> intercept and <i>b</i> is the rate constant for clearance. Data shown are mean ± standard deviation for five mice per dose per time point. Half-life (t_1/2) values calculated from the elimination rate constant (<i>b</i>) derived from the model fitting were similar for all three doses of CPS. The results demonstrate that CPS is eliminated rapidly from serum with a half-life of 4 hours, 4.4 hours, or 2.9 hours for the doses of 100 μg, 20 μg, or 4 μg CPS, respectively.</p

Excreted CPS detection by AMD^™ LFI.

<p>A urine sample from a CPS-treated mouse was serially diluted in mouse control urine to the indicated CPS concentrations. Each concentration of the urine sample then was tested with AMD^™ LFI. The tests were assessed by four examiners in a randomized, semi-blinded manner (panel A), and by using a lateral flow reader (panel B). The results demonstrated that AMD^™ LFI could detect excreted CPS as low as 0.2 ng/mL.</p

Organ distribution of CPS.

<p>Mice were intravenously injected with 100 μg CPS per mouse. Internal organs (lungs, liver, spleen, and kidneys) were collected at various time points post-injection. The organs were homogenized in PBS. CPS amount per organ was determined by quantitative sandwich ELISA. The amount of CPS in blood samples was calculated from the CPS concentration in serum as shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005217#pntd.0005217.g001" target="_blank">Fig 1</a>. Data shown are mean ± standard deviation for five mice per time point. The negative values after subtraction of CPS amounts from serum found in each organ were adjusted to zero. The results showed that no significant amount of CPS accumulated in any of the colletced organs.</p

<i>In vivo</i> Distribution and Clearance of Purified Capsular Polysaccharide from <i>Burkholderia pseudomallei</i> in a Murine Model

<div><p><i>Burkholderia pseudomallei</i> is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The “gold standard” for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, <i>B</i>. <i>pseudomallei</i> capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the <i>in vivo</i> fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis.</p></div

Western blot analysis of excreted CPS.

<p>Purified CPS (lane 1) and urine samples (lanes 2–7) were separated on 7.5% SDS-PAGE gels. All samples including purified CPS were incubated with proteinase K at 60°C for 1 hour, followed by boiling for 10 min before loading on the gels. Lanes 2, 3, and 4 were loaded with control urine spiked with CPS and incubated at 37°C for 30 min, 2 hours, and 8 hours, respectively. Lanes 5, 6, and 7 were urine from CPS-injected mice collected at 30 min, 2 hours, and 8 hours post-injection, respectively. The volume of sample loaded into each lane was adjusted to contain an equal amount of CPS, approximately 1 μg/lane. After blotting, membranes were probed with mAb 4C4 (1 μg/mL). Intact CPS was observed in urine samples from CPS-treated mice.</p

LPS binding affinity of each 1A4 subclass mAb analyzed by SPR using an antibody capture analysis approach.

<p>Anti-mouse antibody was covalently immobilized on a CM5 sensor chip. Each subclass of mAb 1A4 was injected individually over the chip surface, followed by injection of various concentrations of LPS (60–8,000 nM). Data shown is representative of three independent experiments with similar results. <b>Panel A</b> illustrates the complex formed on the chip surface. <b>Panel B</b> presents the sensorgrams (<b>top</b>) and steady-state binding analysis (<b>bottom</b>) of each 1A4 subclass variant.</p

SPR analysis of LPS binding of mAbs 1A4 IgG3, IgG1 and IgG2b using a biotinylated LPS-streptavidin capture platform.

<p>Biotinylated LPS was immobilized on a streptavidin (SA) sensor chip. Sensorgrams were generated by injecting different concentrations of mAbs 1A4 IgG3 (10.4–333 nM), 1A4 IgG1(1.7–8 μM) or 1A4 IgG2b (0.1–3.3 μM) over the chip surface. Data shown is representative of three independent experiments with similar results. <b>Panel A,</b> a diagram depicting the antigen-antibody complex (and a proposed antibody-antibody interaction) formed on the chip surface. <b>Panel B</b> presents the sensorgram profile of 1A4 IgG3 (<b>left</b>) and the corresponding steady-state affinity determination (<b>right</b>). The sensorgrams for 1A4 IgG1 and IgG2b are presented in <b>Panels C and D</b>, respectively.</p

Immunoglobulin G subclass switching impacts sensitivity of an immunoassay targeting <i>Francisella tularensis</i> lipopolysaccharide

<div><p>The CDC Tier 1 select agent <i>Francisella tularensis</i> 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 <i>F</i>. <i>tularensis</i> 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.</p></div

Antibody-antibody interaction as determined by direct ELISA.

<p><b>Panel A</b>, the binding interactions between 1A4 IgG3 and each mAb subclass are demonstrated. <b>Panel B</b>, self-association of each subclass of the mAb 1A4 are shown. The experiments were carried out in quadruplicate. Data shown are mean ± standard deviation.</p

Specificity of mAbs 1A4 and FB11.

<p>Inactivated <i>F</i>. <i>tularensis</i> SCHU S4 (lane 1), SCHU S4 ΔwbtI (lacks <i>O</i>-antigen, lane 2), LVS (lane 3), <i>F</i>. <i>tularensis</i> subsp. <i>holarctica</i> (lane 4), subsp. <i>novicida</i> (lane 5), <i>F</i>. <i>philomiragia</i> (lane 6), purified <i>B</i>. <i>pseudomallei</i> LPS (lane 7) and <i>E</i>. <i>coli</i> LPS (lane 8) were separated on 12% SDS-PAGE gels and blotted onto nitrocellulose membranes. The membranes then were probed with mAbs 1A4 IgG3 (<b>Panel A</b>) and FB11 (<b>Panel B</b>). The existence of LPS in these samples was demonstrated by Pro-Q emerald 300 LPS staining (<b>Panel C</b>).</p