Gd-containing conjugated polymer nanoparticles: bimodal nanoparticles for fluorescence and MRI imaging.

Aqueous bifunctional semiconductor polymer nanoparticles (SPNs), approximately 30 nm in diameter (as measured from electron microscopy), were synthesised using hydrophobic conjugated polymers, amphiphilic phospholipids and a gadolinium-containing lipid. Their fluorescence quantum yields and extinction coefficients were determined, and their MRI T1-weighted relaxation times in water were measured. The bimodal nanoparticles were readily taken up by HeLa and murine macrophage-like J774 cells as demonstrated by confocal laser scanning microscopy, and were found to be MRI-active, generating a linear relationship between T1-weighted relaxation rates and gadolinium concentrations The synthesis is relatively simple, and can easily result in milligrams of materials, although we fully expect scale-up to the gram level to be easily realised.

Investigating the use of protein saver cards for storage and subsequent detection of bovine anti-Brucella abortus smooth lipopolysaccharide antibodies and gamma interferon.

Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature.

Liposomal delivery of p-ialB and p-omp25 DNA vaccines improves immunogenicity but fails to provide full protection against B. melitensis challenge.

BACKGROUND: We have previously demonstrated protective efficacy against B. melitensis using formulations of naked DNA vaccines encoding genes ialB and omp25. The present study was undertaken to further understand the immune response generated by the protective vaccination regimens and to evaluate cationic liposome adsorption as a delivery method to improve vaccine utility. METHODS: The protective efficacy and immunogenicity of vaccines delivered as four doses of naked DNA, a single dose of naked DNA or a single dose of DNA surface adsorbed to cationic liposomes were compared using the BALB/c murine infection model of B. melitensis. Antigen-specific T cells and antibody responses were compared between the various formulations. RESULTS: The four dose vaccination strategy was confirmed to be protective against B. melitensis challenge. The immune response elicited by the various vaccines was found to be dependent upon both the antigen and the delivery strategy, with the IalB antigen favouring CD4+ T cell priming and Omp25 antigen favouring CD8+. Delivery of the p-ialB construct as a lipoplex improved antibody generation in comparison to the equivalent quantity of naked DNA. Delivery of p-omp25 as a lipoplex altered the profile of responsive T cells from CD8+ to CD4+ dominated. Under these conditions neither candidate delivered by single dose naked DNA or lipoplex vaccination methods was able to produce a robust protective effect. CONCLUSIONS: Delivery of the p-omp25 and p-ialB DNA vaccine candidates as a lipoplex was able to enhance antibody production and effect CD4+ T cell priming, but was insufficient to promote protection from a single dose of either vaccine. The enhancement of immunogenicity by lipoplex delivery is a promising step toward improving the practicality of these two candidate vaccines, and suggests that this lipoplex formulation may be of value in situations where improvements to CD4+ responses are required. However, in the case of Brucella vaccine development it is suggested that further modifications to the candidate vaccines and delivery strategies will be required in order to deliver sustained protection.

Time-resolved fluorescent resonance energy transfer assay for simple and rapid detection of anti-Brucella antibodies in ruminant serum samples.

Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases.

Competitive electrochemiluminescence wash and no-wash immunoassays for detection of serum antibodies to smooth Brucella strains.

Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISA(SP)SS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases.

A new homogeneous assay for high throughput serological diagnosis of brucellosis in ruminants.

The control and eradication of brucellosis is highly desirable but heavily resource intensive as high throughput serological testing is required. The aim of this study was to meet the needs of high throughput screening laboratories involved in this process through the development of a new assay. An existing cELISA used for the serodiagnosis of brucellosis in ruminants was converted to an AlphaLISA homogenous proximity based assay. This assay requires no separation steps and can be performed in low volume microtitre format. The Brucella AlphaLISA was validated on a panel of bovine, ovine and caprine sera from infected and uninfected animals. The diagnostic sensitivities (>96%) and specificities (>98%) obtained compared well to those from cELISA, iELISA and FPA performed on the same samples. The AlphaLISA met the testing criteria set for ELISAs as defined by the OIEELISA standards and had an analytical sensitivity similar to that of the parent cELISA. The method was also used on a small panel of serum samples from cattle that were experimentally infected with Yersinia enterocolitica O:9. Some false positive reactions were obtained as was also the case with results from FPA, iELISA, cELISA, CFT and SAT. Despite this, the methodological advantages of the AlphaLISA mean that this assay is well suited to high throughput serodiagnosis. This report is the first description of the use of AlphaLISA to detect pathogen specific antibodies. Furthermore, the relative ease with which the cELISA was converted to this platform indicates that this technology is ready to meet the high throughput testing requirements for the diagnosis of many other diseases.

The identification of two protective DNA vaccines from a panel of five plasmid constructs encoding Brucella melitensis 16M genes.

Five candidate genes from the Brucella melitensis 16M genome were selected. Eukaryotic expression plasmids encoding these antigens were constructed and expression was verified in vitro from transfected Cos7 cells. Each vaccine was assessed for protective efficacy in a BALB/c mouse brucellosis infection model. From these experiments two protective DNA vaccines were identified: p-omp25 and p-ialB. The Omp25 antigen (BMEI1249) has previously been studied in terms of Brucella virulence, serodiagnosis and as a protective antigen. However, this study represents the first report of a significant protective effect achieved against B. melitensis 16M challenge using the Omp25 antigen in a DNA vaccine approach. The other protective vaccine identified in this study was p-ialB. The ialB candidate (BMEI1584) was selected based upon its' putative function as an invasion protein which was assigned due to shared identity with the invasion protein B (ialB) of Bartonella bacilliformis. This candidate has not previously been investigated with regard to Brucella virulence or pathogenesis. This study is the first report to identify the Brucella invasion protein B (BMEI1584) as a novel protective antigen for brucellosis.

Type III secretion homologs are present in Brucella melitensis, B. ovis, and B. suis biovars 1, 2, and 3.

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.