Search Results (10)

Valley Fever (VF), caused by fungi in the genus Coccidioides, is a prevalent disease in southwestern and western parts of the United States that affects both humans and animals, such as dogs. Although the immune responses to infection with Coccidioides spp. are not fully characterized, antibody-detection assays are used in conjunction with clinical presentation and radiologic findings to aid in the diagnosis of VF. These assays often use Complement Fixation (CF) and Tube Precipitin (TP) antigens as the main targets of IgG and IgM reactivity, respectively. Our group previously reported evidence of over 800 genes expressed at the protein level in C. posadasii. However, antibody reactivity to the majority of these proteins has never been explored. Using a new, high-throughput screening technology, the Nucleic Acid Programmable Protein Array (NAPPA), we screened serum specimens from dogs against 708 of these previously identified proteins for IgG reactivity. Serum from three separate groups of dogs was analyzed and revealed a small panel of proteins to be further characterized for immuno-reactivity. In addition to CF/CTS1 antigen, sera from most infected dogs showed antibody reactivity to endo-1,3-betaglucanase, peroxisomal matrix protein, and another novel reactive protein, CPSG_05795. These antigens may provide additional targets to aid in antibody-based diagnostics. Full article

For the rapid detection of bacteria in a blood sample, nucleic acid amplification-based assays are believed to be promising. Nevertheless, the nucleic acids released from the dead blood cells or bacteria could affect the assay performance. This highlights the importance of the isolation of live bacteria from blood samples. To address this issue, this study proposes a two-step process. First, a blood sample was treated with the immuno-magnetic microbeads-based separation to remove the majority of blood cells. Second, an optically induced dielectrophoresis (ODEP) microfluidic system with an integrated dynamic circular light image array was utilized to further isolate and purify the live bacteria from the remaining blood cells based on their size difference. In this work, the ODEP microfluidic system was developed. Its performance for the isolation and purification of bacteria was evaluated. The results revealed that the method was able to harvest the live bacteria in a high purity (90.5~99.2%) manner. Overall, the proposed method was proven to be capable of isolating and purifying high-purity live bacteria without causing damage to the co-existing cells. This technical feature was found to be valuable for the subsequent nucleic-acid-based bacteria detection, in which the interferences caused by the nontarget nucleic acids could be eliminated. Full article

Although aluminium-based vaccines have been used for almost over a century, their mechanism of action remains unclear. It is established that antigen adsorption to the adjuvant facilitates delivery of the antigen to immune cells at the injection site. To further increase our understanding of aluminium-based vaccines, it is important to gain additional insights on the interactions between the aluminium and antigens, including antigen distribution over the adjuvant particles. Immuno-assays can further help in this regard. In this paper, we evaluated how established formulation strategies (i.e., sequential, competitive, and separate antigen addition) applied to four different antigens and aluminium oxyhydroxide, lead to formulation changes over time. Results showed that all formulation samples were stable, and that no significant changes were observed in terms of physical-chemical properties. Antigen distribution across the bulk aluminium population, however, did show a maturation effect, with some initial dependence on the formulation approach and the antigen adsorption strength. Sequential and competitive approaches displayed similar results in terms of the homogeneity of antigen distribution across aluminium particles, while separately adsorbed antigens were initially more highly poly-dispersed. Nevertheless, the formulation sample prepared via separate adsorption also reached homogeneity according to each antigen adsorption strength. This study indicated that antigen distribution across aluminium particles is a dynamic feature that evolves over time, which is initially influenced by the formulation approach and the specific adsorption strength, but ultimately leads to homogeneous formulations. Full article

In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15–115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications. Full article

An immuno-separated assay for ochratoxin A detection coupled with a nano-affinity cleaning up for LC-confirmation was developed. Firstly, ochratoxin A was modified to quantum dot beads for immuno-fluorescent reporters. Secondly, Fe₃O₄ magnetic nanoparticles were conjugated with protein G for immuno-magnetic adsorbents. The immuno-separation of fluorescent reporters by magnetic adsorbents could be completed by ochratoxin A, so the fluorescent reporters released from the immune complex indicate a linear correlation with the concentration of ochratoxin A. Furthermore, the immuno-separated ochratoxin A can be eluted from magnetic adsorbent for LC-conformation. The optimized assay showed results as follows: the quantitative range of the immuno-separated assay was 0.03–100 ng mL⁻¹ of ochratoxin A. The recoveries for spiked samples ranged from 78.2% to 91.4%, with the relative standard deviation (RSD) being 11.9%~15.3%. Statistical analysis indicated no significant difference between the HPLC-FLD results based on commercial affinity column and by nano-affinity cleaning up. Full article

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9⁺, CD63⁺, and CD81⁺ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9⁺/CD63⁺/CD81⁺EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen. Full article

Compression wood is a type of reaction wood formed on the underside of softwood stems when they are tilted from the vertical and on the underside of branches. Its quantification is still a matter of some scientific debate. We developed a new technique that has the potential to do this based on the higher proportions of (1→4)-β-d-galactans that occur in tracheid cell walls of compression wood. Wood was milled, partially delignified, and the non-cellulosic polysaccharides, including the (1→4)-β-d-galactans, extracted with 6 M sodium hydroxide. After neutralizing, the solution was serially diluted, and the (1→4)-β-d-galactans determined by an immuno-dot assay using the monoclonal antibody LM5, which specifically recognizes this polysaccharide. Spots were quantified using a dilution series of a commercially available (1→4)-β-d-galactan from lupin seeds. Using this method, compression and opposite woods from radiata pine (Pinus radiata) were easily distinguished based on the amounts of (1→4)-β-d-galactans extracted. The non-cellulosic polysaccharides in the milled wood samples were also hydrolysed using 2 M trifluoroacetic acid followed by the separation and quantification of the released neutral monosaccharides by high performance anion exchange chromatography. This confirmed that the compression woods contained higher proportions of galactose-containing polysaccharides than the opposite woods. Full article

Lab-on-a-chip immuno assays utilizing superparamagnetic beads as labels suffer from the fact that the majority of beads pass the sensing area without contacting the sensor surface. Different solutions, employing magnetic forces, ultrasonic standing waves, or hydrodynamic effects have been found over the past decades. The first category uses magnetic forces, created by on-chip conducting lines to attract beads towards the sensor surface. Modifications of the magnetic landscape allow for additional transport and separation of different bead species. The hydrodynamic approach uses changes in the channel geometry to enhance the capture volume. In acoustofluidics, ultrasonic standing waves force µm-sized particles onto a surface through radiation forces. As these approaches have their disadvantages, a new sensor concept that circumvents these problems is suggested. This concept is based on the granular giant magnetoresistance (GMR) effect that can be found in gels containing magnetic nanoparticles. The proposed design could be realized in the shape of paper-based test strips printed with gel-based GMR sensors. Full article

Simple immuno-magnetic separation tandem fluorescent probes based on quantum dots-antibody (QDs-Ab) were developed to detect Salmonella with sensitivity of 500 cfu mL⁻¹. With two monoclonal antibodies, which recognize different antigenic determinant on the surface of Salmonella, we prepared antibody-coated magnetic nanoparticles (MNPs) and conjugates of QDs-Ab. The immune-magnetic beads were verified with high enrichment efficiency for Salmonella (90%). A sandwich structure formed if the Salmonella solution was mixed together with immune-beads and QDs-Ab, and the fluorescent single from QDs was related to the amount of Salmonella. A linear response between fluorescence intensity and various concentrations of Salmonella (2.5 × 10³ to 1.95 × 10⁸ cfu mL⁻¹) were observed with this proposed method. The total assay time for Salmonella was 30 min, and no cross-reaction to other microbial strains, such as Staphylococcus aureus, Escherichia coli (E. coli) and Escherichia coli O157:H7 (E. coli O157:H7), were found using this detection system. All our results showed that the simple homogeneous immunoassay could be applied in Salmonella screening without time-consuming extra-enrichment of bacteria. Full article

Acoustic wave biosensors are a real-time, label-free biosensor technology, which have been exploited for the detection of proteins and cells. One of the conventional biosensor approaches involves the immobilization of a monolayer of antibodies onto the surface of the acoustic wave device for the detection of a specific analyte. The method described within includes at least two immobilizations of two different antibodies onto the surfaces of two separate acoustic wave devices for the detection of several analogous analytes. The chemical specificity of the molecular recognition event is achieved by virtue of the extremely high (nM to pM) binding affinity between the antibody and its antigen. In a standard ELISA (Enzyme-Linked ImmunoSorbent Assay) test, there are multiple steps and the end result is a measure of what is bound so tightly that it does not wash away easily. The fact that this “gold standard” is very much not real time, masks the dance that is the molecular recognition event. X-Ray Crystallographer, Ian Wilson, demonstrated more than a decade ago that antibodies undergo conformational change during a binding event[1, 2]. Further, it is known in the arena of immunochemistry that some antibodies exhibit significant cross-reactivity and this is widely termed antibody promiscuity. A third piece of the puzzle that we will exploit in our system of acoustic wave biosensors is the notion of chemical orthogonality. These three biochemical constructs, the dance, antibody promiscuity and chemical orthogonality will be combined in this paper with the notions of Int. J. Mol. Sci. 2008, 9 155 in-phase (I) and quadrature (Q) signals from digital radio to manifest an approach to molecular recognition that allows a level of discrimination and analysis unobtainable without the aggregate. As an example we present experimental data on the detection of TNT, RDX, C4, ammonium nitrate and musk oil from a system of antibody-coated acoustic wave sensors. Full article