Microbead Assay Research Articles

Evaluation of the Transforming Growth Factor-β Activity in Normal and Dry Eye Human Tears by CCL-185 Cell Bioassay

To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.

Performance evaluation of microbead and ELISA assays for follicular G-CSF: a non-invasive biomarker of oocyte developmental competence for embryo implantation

G-CSF in individual follicular fluids correlates with the potential of the corresponding embryo to result in a live birth after transfer in IVF. To evaluate the requirements for routine follicular fluid G-CSF quantification, we compared follicular fluid G-CSF measurements made with two multiplexed microbead assays purchased from Bio-Rad Laboratories and R&D Systems, and a commercial G-CSF ELISA (R&D Systems). Individual follicular fluids (n=139) associated with transferred embryos were analysed to determine cytokine profile and the fate of each transferred embryo was recorded. The effect of multiplexing as well as comparison of the respective performances of the microbead assay with a flow cytometry assay was explored. Multivariable logistic regression analysis was performed and receiver operating characteristic (ROC) analysis was used to determine the performance and sensitivity/specificity of each method for individual follicular fluids. Covariate factors known to influence IVF outcome such as age, serum oestradiol and embryo score were systematically integrated in each analysis. The quantification of follicular fluid G-CSF using microbead assay methodologies, but not ELISA, yielded results showing the utility of follicular fluid G-CSF as a biomarker predictive of a successful delivery (Au(roc): 0.77 [0.68-0.84] (p=0.003) and 0.75 [0.66-0.82] (p=0.004) for Bio-Rad and R&D Systems microbead assays respectively), whereas follicular fluid G-CSF values quantified by ELISA were not predictive (Au(roc):0.61 [0.52-0.70] p=0.84). Microbead assay and flow cytometry appeared similarly efficient for quantifying follicular fluid G-CSF and multiplex versus single-plex assays did not influence the reliability of quantification.

OL-051 Molecular-genetic analysis of drug-resistant Mycobacterium tuberculosis strains in St. Petersburg (Russia)

cardiac puncture. The antibodies were confirmed by Western blots and Multiplex Microbead Assay (MMA). The best humoral response was shown by hspx-pND vaccinated animals both on Western blots and MMA. Fairly positive response was obtained in animals vaccinated with esat6-pND14 and (ag85 a, b and c)-pND vaccines. Whereas undetectable level of antibodies from cfp10-pND vaccinated animals were produced. In general the results of this study are promising but need more animal studies before the constructs go to clinical trials.

Multifunctional Dendrimer-templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection.

Dendrimers, with their well-defined globular shape and a high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. Synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on to the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich type ELISA to detect IL-6 and IL-1β, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13 pg ml-1 for IL-6 luminol detection and 1.15 pg ml-1 for IL-1β TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from normal (non-pregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors and field effect biosensors.

Selectin Mechanokinetics and Two-Dimensional Bond Formation Determine and are Reported by Nano-Motion Dynamic Patterns

Binding between surface-tethered proteins at cellular interfaces has been considered two-dimensional because of the restricted motion of the two binding partners. Two-dimensional protein interactions between cells are critical for many biological processes, such as leukocyte vascular adhesion via selectins. Experimental measurements have yielded data on the kinetics of selectin bond formation and dissociation. Additionally, computational methods have been employed to integrate molecular and cellular properties to elucidate the factors that influence the dynamics of selectin-mediated rolling. Simulation methods focused on biomolecular properties promise to yield additional novel insights into the molecular component of adhesion with the assistance of measurements from improved assays. We performed an in silico investigation on the effects of the kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, two-dimensional bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Statistics describing the motion patterns tied simulated motions to experimentally reported quantities. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve pause times observed in microbead assays. Notably, rebinding events were enhanced by the reduced separation distance following initial sphere capture. The result demonstrates vertical transport can contribute to an enhancement in the apparent bond formation rate. The result also suggests a new mechanism that may be important for the rebinding events characteristic of stable leukocyte rolling. When selectin receptor and ligand are restricted to small, two-dimensional interaction zones during rolling, the resultant wobble was found to be dependent on the confinement model used. Insight into two-dimensional bond formation gained from flow cell assays might also therefore be important to understand processes involving extended cellular interactions, such as immunological synapse formation.

Nano-motion Dynamics are Determined by Surface-Tethered Selectin Mechanokinetics and Bond Formation

The interaction of proteins at cellular interfaces is critical for many biological processes, from intercellular signaling to cell adhesion. For example, the selectin family of adhesion receptors plays a critical role in trafficking during inflammation and immunosurveillance. Quantitative measurements of binding rates between surface-constrained proteins elicit insight into how molecular structural details and post-translational modifications contribute to function. However, nano-scale transport effects can obfuscate measurements in experimental assays. We constructed a biophysical simulation of the motion of a rigid microsphere coated with biomolecular adhesion receptors in shearing flow undergoing thermal motion. The simulation enabled in silico investigation of the effects of kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, surface-constrained bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Simulations recreated the jerky, discrete stop-and-go motions observed in P-selectin/PSGL-1 microbead assays with physiologic ligand densities. Motion statistics tied detailed simulated motion data to experimentally reported quantities. New deductions about biomolecular function for P-selectin/PSGL-1 interactions were made. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve bond lifetimes observed in microbead assays. Initial, capturing bond formation effectively occurred across the entire molecular contour length. However, subsequent rebinding events were enhanced by the reduced separation distance following the initial capture. The result demonstrates that vertical transport can contribute to an enhancement in the apparent bond formation rate. A detailed analysis of in silico motions prompted the proposition of wobble autocorrelation as an indicator of two-dimensional function. Insight into two-dimensional bond formation gained from flow cell assays might therefore be important to understand processes involving extended cellular interactions, such as immunological synapse formation. A biologically informative in silico system was created with minimal, high-confidence inputs. Incorporating random effects in surface separation through thermal motion enabled new deductions of the effects of surface-constrained biomolecular function. Important molecular information is embedded in the patterns and statistics of motion.

Quantitative considerations for suspension array assays

A novel format of a multiplex microbead assay that is capable of accurately and reproducibly measuring small changes in 10 8 copies of a nucleic acid is reported herein. The dynamic range of the assay was 32-fold greater than conventional multiplex microbead assays. The assay was modeled mathematically which enabled the accuracy of DNA quantification to be formally analysed using a logical theoretical framework. The assay was also evaluated on RNA extracts taken from stimulated macrophages, to demonstrate that changes in gene expression can be accurately monitored in a biological system. We believe the reproducibility and accuracy of the assay presented herein will be useful for high-throughput, multiplex sensitive detection of nucleic acids over a large dynamic range in a research or diagnostic setting.

Systemic and Local Interleukin-17 and Linked Cytokines Associated with Sjögren’s Syndrome Immunopathogenesis

Recently recognized as a distinct CD4(+) T helper (Th) lineage, Th17 cells have been implicated in host responses to infections and in pathogenesis associated with autoimmune diseases. This cytokine is implicated in primary Sjögren's syndrome (pSS) immunopathology because of the increased levels of circulating interleukin (IL)-17 in pSS. Plasma and minor salivary glands (MSGs) from patients with pSS were therefore evaluated for CD4(+) T cells, T regulatory cells, IL-17, and supporting cytokines by immunohistochemistry, RT-PCR, and microbead assays. MSGs from pSS patients contain IL-17-expressing cells as a dominant population within inflammatory lesions. IL-17 protein expression progressively increased with higher biopsy focus scores (P < 0.0001), in parallel with detection by RT-PCR. Transforming growth factor-beta, IL-6 and IL-23, which are requisite promoters of Th17 differentiation, were found in abundance compared with the amounts in control tissues. Although transforming growth factor-beta is also a pivotal differentiation factor for immunosuppressive Foxp3(+) T regulatory cells (Tregs), an increase in Foxp3(+) Tregs was evident in biopsy specimens with mild and moderate inflammation but this increase was disproportionate to escalating pro-inflammatory Th17 populations in advanced disease. Furthermore, the Th17-centric cytokines IL-17, IL-6, IL-23, and IL-12 were significantly elevated in pSS plasma. These data identify a profusion of IL-17-generating cells and supporting cytokines within diseased pSS MSGs without a compensatory increase in immunomodulatory Tregs; this imbalance seems to foster a pathogenic milieu that may be causative and predictive of infiltrative injury and amenable to therapeutic intervention.

Flexible Acoustic Particle Manipulation Device with Integrated Optical Waveguide for Enhanced Microbead Assays

Realisation of a device intended for the manipulation and detection of bead-tagged DNA and other bio-molecules is presented. Acoustic radiation forces are used to manipulate polystyrene micro-beads into an optical evanescent field generated by a laser pumped ion-exchanged waveguide. The evanescent field only excites fluorophores brought within approximately 100 nm of the waveguide, allowing the system to differentiate between targets bound to the beads and those unbound and still held in suspension. The radiation forces are generated in a standing-wave chamber that supports multiple acoustic modes, permitting particles to be both attracted to the waveguide surface and also repelled. To provide further control over particle position, a novel method of switching rapidly between different acoustic modes is demonstrated, through which particles are manipulated into an arbitrary position within the chamber. A novel type of assay is presented: a mixture of streptavidin coated and control beads are driven towards a biotin functionalised surface, then a repulsive force is applied, making it possible to determine which beads became bound to the surface. It is shown that the quarter-wave mode can enhance bead to surface interaction, overcoming potential barriers caused by surface charges. It is demonstrated that by measuring the time of flight of a microsphere across the device the bead size can be determined, providing a means of multiplexing the detection, potentially detecting a range of different target molecules, or varying bead mass.

Analysis of a high‐throughput HLA antibody screening assay for use with platelet donors

Passive infusion of HLA antibodies has been implicated in transfusion reactions. A rapid, inexpensive method of screening blood donors for HLA antibodies might reduce the incidence of reactions. A high-throughput microbead-flow analyzer HLA antibody detection technique was compared with an enzyme-linked immunosorbent assay (ELISA) method. Ninety-six apheresis platelet (PLT) donors were tested for antibodies to Class I and II HLA antigens with mixed-antigen microbead-flow analyzer and ELISAs. For both assays, samples reactive in the mixed-antigen assay were tested with a panel-reactive antibody (PRA) assay. Samples reactive in both the mixed-antigen and the PRA assays were considered positive. In the mixed-antigen microbead assay, 46 (48%) samples were reactive to Class I antigens and 20 (21%) to Class II. Further testing in the microbead PRA assay revealed that 34 (35%) had antibodies to Class I antigens, 18 (19%) to Class II, and 42 (44%) to either Class I or Class II. Class I antibodies were present in 56 percent of females and 36 percent of males. In the mixed-antigen ELISA, 4 samples were reactive with Class I antigens, 4 with Class II antigens, and 5 with Class I or Class II. All 5 reactive samples were also reactive in the ELISA PRA assay and were from females. The microbead assay was more sensitive than the ELISA and detected antibodies in a large proportion of donors. Samples reactive in the mixed-antigen microbead assay should be confirmed by a second assay before concluding that antibodies are present.

Heteroduplex analysis using flow cytometric microbead assays to detect deletions, insertions, and single‐strand lesions

We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin- and dye labeled reaction products are immobilized on microbeads and the homo- and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero- from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single-strand lesions formed at hypersensitive sites in response to apoptotic stimuli.

Efficient factor VIII affinity purification using a small synthetic ligand.

Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. Peptide ligands were screened for binding of (125)I-plasma-derived-FVIII (pdFVIII) in a microbead assay. A selected ligand-coated Toyopearl resin was then used for pdFVIII purification from cell-conditioned Delbucco's modified Eagle's medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV-MZ-6 cells was assayed. A high-affinity octapeptidic FVIII ligand was modified into the small, highly stable and non-toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4-coated Toyopearl resin, pdFVIII was isolated from cell-conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. Ligand L4 offers a valuable alternative to antibody-based procedures for laboratory and industrial production. Its synthesis by established solid-phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.

DNA typing by microbead arrays and PCR‐SSP: apparent false‐negative or ‐positive hybridization or amplification signals disclose new HLA‐B and ‐DRB1 alleles

Human leukocyte antigen (HLA) typing by polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) hybridization on solid phase (microbead assay) or polymerase chain reaction-sequence-specific primers (PCR-SSP) requires interpretation softwares to detect all possible allele combinations. These programs propose allele calls by taking into account false-positive or false-negative signal(s). The laboratory has the option to validate typing results in the presence of strongly cross-reacting or apparent false-negative signals. Alternatively, these seemingly aberrant signals may disclose novel variants. We report here four new HLA-B (B*5620 and B*5716) and HLA-DRB1 alleles (DRB1*110107 and DRB1*1474) that were detected by apparent false-negative or -positive hybridization or amplification patterns, and ultimately resolved by sequencing. To avoid allele misassignments, a comprehensive evaluation of acquired data as documented in a quality assurance system is therefore required to confirm unambiguous typing interpretation.

A Novel PF4-Heparin Microbead Assay and Plasma Protein Biomarker Profiling for Improved Diagnosis of Heparin Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening, immune-mediated complication of heparin administration resulting in a hypercoagulable state. The diagnosis is made using clinical scoring systems with confirmatory laboratory testing. However, current diagnostic criteria may lack predictive value for HIT-associated thrombosis and need for alternative anticoagulation. Therefore, there is a need to develop diagnostic methods that will have a better predictive value for HIT diagnosis. The pathogenesis of HIT involves platelet-activating antibodies that recognize platelet factor 4 (PF4) -heparin complex, and may also involve dysregulation of inflammatory mediators and growth factors from platelets and endothelial cells. Luminex multiplex microbead assay allows for the simultaneous detection of multiple serum analytes. The aim of this study is to develop a multiplex method to detect anti-PF4-heparin antibodies and to generate profiles of cytokines, chemokines, growth factors and adhesion molecules with better predictive value for the diagnosis of HIT than current diagnostic methods. In combination with measurement of antibodies to (PF4)-heparin complex, such a panel is expected to enhance the accuracy of HIT diagnosis. To develop the anti-PF4-heparin IgG microbead assay, carboxylated Luminex microbeads were coated at various concentrations with the optimal molar ratio of the PF4/polyvinyl sulfonate (PF4/PVS) complex, the antigenic determinant from the GTI ELISA kit, (provided by GTI, Waukesha WI). The anti-PF4-heparin microbead assay was standardized using plasma samples from patients and healthy controls. Furthermore, we compared the anti-PF4-heparin microbead assay to traditional commercial HIT antibody ELISA kits from GTI, Diagnostica Stago and Aniara. To identify potential plasma biomarkers for HIT, measurements were made on plasma levels of 63 cytokines, chemokines, growth factors and adhesion molecules in patients with and without HIT (as defined by the Chong's score) and healthy controls using commercial multiplex detection panels (Bio-Rad, Upstate, Linco). Multiplex data were analyzed using multivariate statistical methods and compared with their respective clinical scores. Preliminary anti-PF4-heparin microbead assay results show an overall concordance rate of 80% with GTI (Polyclonal) ELISA; 91% with Diagnostica Stago (Polyclonal); 78% with Aniara Polyclonal and 76% with Aniara IgG ELISA. The anti-PF4-heparin microbead assay results also show good correlation with clinical determination of HIT disease status as determined by Chong's scoring (72% sensitivity, and 58% specificity). Preliminary biomarker profiling suggest that Il-2Ra, b-NGF, GRO-a, TNF-b, PDGF AB/BB, PDGF AA, and s-ICAM1 are potentially useful markers in further analysis for distinguishing HIT status. We have identified these candidate biomarkers to evaluate in a larger number of patient samples with suspected HIT. Our data show that our novel microbead assay for anti-PF4-heparin antibodies correlate well with the existing conventional ELISA results, and also correlate with HIT diagnosis as determined by the Chong's score. Furthermore, we have identified a panel of biomarkers that could be useful for improved diagnosis and prognosis of HIT, and will establish a basis for further understanding the pathophysiology of this coagulation disorder.

Blood Levels of Donor-Specific Human Leukocyte Antigen Antibodies After Renal Transplantation: Resolution of Rejection in the Presence of Circulating Donor-Specific Antibody

Accommodation to antibody is an important mechanism in successful ABO-incompatible transplantation, but its importance in human leukocyte antigen (HLA) antibody-incompatible transplantation is less clear, as sensitive techniques facilitating daily measurement of donor-specific HLA antibodies (DSAs) have only recently been developed. We report 24 patients who had HLA antibody-incompatible kidney transplantation (21 living donors, 3 deceased), 21 of whom had pretransplant plasmapheresis. Eight had positive complement-dependent cytotoxic (CDC) crossmatch (XM) pretransplant plasmapheresis, nine had positive flow cytometric (FC) XM, and seven had DSA detectable by microbead analysis only. After transplant, DSA levels were monitored closely with microbead assays. Rejection occurred in five of eight (62.5%) CDC-positive cases, in three of nine (33%) FC-positive cases, and in two of seven (29%) of microbead-only cases at a median of 6.5 days after transplantation. Resolution occurred at a median of 15 days after transplantation, in 8 of 10 cases when the microbead level of DSA had median fluorescence intensity (MFI) >2000 U, in 6 of 10 when the microbead MFI >4000 U. In 8 of 10 cases, the microbead MFI at the time of resolution was greater than at the onset. DSA did not always cause clinical rejection. In five cases with a posttransplant DSA peaking at MFI >2000 U on microbead assay, rejection did not occur. These data suggest that the dominant method of successful transplantation was function of the transplant in the presence of circulating DSA, and they also define the period during which this occurred.

Phosphoproteomic approaches to elucidate cellular signaling networks

Protein phosphorylation is crucial in the regulation of signaling pathways that control various biological responses. Recent progress in diverse methodologies to investigate protein phosphorylation in complex biological samples has resulted in more rapid, detailed and quantitative analyses of signaling networks. In particular, advances in mass spectrometry (MS) have enabled the identification and quantification of thousands of both known and novel phosphorylation sites. Initial MS-based information can be complemented with a variety of recently developed and improved phosphoproteomic techniques. These include multiplexed microbead or kinase activity assays, flow cytometry based single-cell analysis, protein microarrays and interaction studies. The combination of multiple approaches, coupled with phenotypic response measurements, computational modeling and biochemical manipulations, will ultimately reveal the mechanistic regulation of signaling networks.

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.

Simultaneous Serodetection of 10 Highly Prevalent Mouse Infectious Pathogens in a Single Reaction by Multiplex Analysis

Under current practices of mouse colony maintenance, sera from mice are analyzed for antibodies against several widespread infectious pathogens by conventional immunoassays, generally enzyme-linked immunosorbent assay (ELISA). To test for multiple agents, these methods consume large volumes of mouse serum and are laborious and time-consuming. More efficient immunoassays, using small amounts of sample, are therefore needed. Accordingly, we have developed a novel multiplex diagnostic system that employs fluorescent microbeads, coated with purified antigens, for simultaneous serodetection of 10 mouse infectious agents. Individually identifiable, fluorescent microbeads were coated with antigens from Sendai virus, mouse hepatitis virus, Theiler's mouse encephalomyelitis virus/GDVII strain, mouse minute virus, mouse cytomegalovirus, respiratory enteric orphan virus (Reo-3 virus), mouse parvovirus, calf rotavirus for epizootic diarrhea virus of infant mice, vaccinia virus for ectromelia virus, and Mycoplasma pulmonis. Standard sera, singly positive for antibodies to individual infectious agents, were generated by inoculation of BALB/cj and C57BL/6j mice. Sera from these experimentally infected mice, as well as sera from naturally infected mice, were analyzed using a mixture of microbeads coated with antigens of the 10 infectious agents listed above. Results demonstrated that the multiplex assay was at least as sensitive and specific as ELISA for serodetection. Importantly, the multiplex assay required only 1 microliter of serum for simultaneous serodetection of the 10 mouse infectious agents in one reaction vessel. Thus, this multiplex microbead assay is a reliable, efficient, and cost-effective diagnostic modality that will impact serosurveillance of mice used in research.

RecombinantHelicobacter bilisProtein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated to purified, H. bilis-specific, recombinant proteins P167C and P167D and bacterial membrane extracts from H. bilis and Helicobacter hepaticus. For detecting H. bilis infection in the microbead multiplex assay, P167C and P167D provided significantly higher sensitivities (94 and 100%, respectively) and specificities (100 and 95%, respectively) than membrane extract (78% sensitivity and 65% specificity). Microbead multiplex assay results were validated by enzyme-linked immunosorbent assay. Purified recombinant proteins showed low batch-to-batch variation; this feature allows for ease of quality control, assay robustness, and affordability. Thus, recombinant antigens are highly suitable in the multiplex microbead assay format for serodetection of H. bilis infection.

Emerging Technologies for Mycotoxin Detection

The history of the development of analytical methods for detecting fungal toxins is rich and varied. Method development has followed a process somewhat akin to Darwinian evolution: methods are selected based upon the characteristics most desirable to the analyst. Typically, this has lead to the development of accurate and sensitive methods for their detection, with a recurring emphasis on improving the speed and lowering the costs of the assays. Like evolution, there have been radical developments, incremental developments, and techniques that have fallen from favor only to be rediscovered. This review focuses on recent developments in technologies for detection of mycotoxins, with a particular emphasis on the myriad forms of biosensors that have begun to appear. Specifically, recent development in evanescent wave technologies (surface plasmon resonance, fiber optic sensors), lateral flow and dipstick devices, fluorescence polarization and time‐resolved fluorescence, microbead assays, and capillary electr...