Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA), the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation), whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis

Genetic and pathological characteristics of <i>Cryptococcus gattii</i> and <i>Cryptococcus neoformans</i> var. <i>neoformans</i> from meningoencephalitis in autochthonous goats and mouflons, Sardinia, Italy

In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain

Sensitivity of LFA for detection of purified poly-γ-D-glutamic acid (PGA) in pooled normal rabbit plasma and human serum.

<p>The top line is a control indicating proper flow of the lateral flow device. The bottom line is the test line for detection of PGA. Results of visual evaluation by four individuals are reported as negative (-) or positive (+) below each assay; (*) denotes a result that was considered weak, but still deemed positive by at least 3 out of 4 evaluators.</p

Detection and Quantification of the Capsular Polysaccharide of Burkholderia pseudomallei in Serum and Urine Samples from Melioidosis Patients.

Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease common in Southeast Asia and northern Australia. Melioidosis often presents with nonspecific symptoms and has a fatality rate of upwards of 70% when left untreated. The gold standard for diagnosis is culturing B. pseudomallei from patient samples. Bacterial culture, however, can take up to 7 days, and its sensitivity is poor, at roughly 60%. The successful administration of appropriate antibiotics is reliant on rapid and accurate diagnosis. Hence, there is a genuine need for new diagnostics for this deadly pathogen. The Active Melioidosis Detect (AMD) lateral flow immunoassay (LFI) detects the capsular polysaccharide (CPS) of B. pseudomallei. The assay is designed for use on various clinical samples, including serum and urine; however, there are limited data to support which clinical matrices are the best candidates for detecting CPS. In this study, concentrations of CPS in paired serum and urine samples from melioidosis patients were determined using a quantitative antigen capture enzyme-linked immunosorbent assay. In parallel, samples were tested with the AMD LFI, and the results of the two immunoassays were compared. Additionally, centrifugal concentration was performed on a subset of urine samples to determine if this method may improve detection when CPS levels are initially low or undetectable. The results indicate that while CPS levels varied within the two matrices, there tended to be higher concentrations in urine. The AMD LFI detected CPS in 40.5% of urine samples, compared to 6.5% of serum samples, suggesting that urine is a preferable matrix for point-of-care diagnostic assays. IMPORTANCE Melioidosis is very challenging to diagnose. There is a clear need for a point-of-care assay for the detection of B. pseudomallei antigen directly from patient samples. The Active Melioidosis Detect lateral flow immunoassay detects the capsular polysaccharide (CPS) of B. pseudomallei and is designed for use on various clinical samples, including serum and urine. However, there are limited data regarding which clinical matrix is preferable for the detection of CPS. This study addresses this question by examining quantitative CPS levels in paired serum and urine samples and relating them to clinical parameters. Additionally, centrifugal concentration was performed on a subset of urine samples to determine whether this might enable the detection of CPS in samples in which it was initially present at low or undetectable levels. These results provide valuable insights into the detection of CPS in patients with melioidosis and suggest potential ways forward in the diagnosis and treatment of this challenging disease

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

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

Optimization of an Antibody Microarray Printing Process Using a Designed Experiment

Antibody microarrays have proven useful in immunoassay-based point-of-care diagnostics for infectious diseases. Noncontact piezoelectric inkjet printing has advantages to print antibody microarrays on nitrocellulose substrates for this application due to its compatibility with sensitive solutions and substrates, simple droplet control, and potential for high-capacity printing. However, there remain real-world challenges in printing such microarrays, which motivated this study. The effects of three concentrations of capture antibody (cAb) reagents and nozzle hydrostatic pressures were chosen to investigate three responses: the number of printed membrane disks, dispensing performance, and microarray quality. Printing conditions were found to be most ideal with 5 mg/mL cAb and a nozzle hydrostatic pressure near zero, which produced 130 membrane disks in a single print versus the 10 membrane disks per print before optimization. These results serve to inform efficient printing of antibody microarrays on nitrocellulose membranes for rapid immunoassay-based detection of infectious diseases and beyond

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

Sequence alignments of mAb 1A4 IgG3, IgG1 and IgG2b variable regions.

<p>The nucleotide sequences of mAbs 1A4 IgG3, IgG1 and IgG2b variable regions were analyzed using IMGT/V-Quest (<a href="http://www.imgt.org" target="_blank">www.imgt.org</a>). The resulting amino acid sequences of heavy chain (<b>Panel A</b>) and light chain (<b>Panel B</b>) framework regions (FR) 1, 2, and 3 and complementarity-determining regions (CDR) 1, 2, and 3 are displayed. The grey highlights indicate the amino acid residues of CDRs.</p