Multiplexed particle-based flow cytometric assays

Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent development of particle-based flow cytometric assays has raised hopes that many of these limitations can be overcome. The technology utilizes microspheres as the solid support for a conventional immunoassay, affinity assay or DNA hybridization assay which are subsequently analyzed on a flow cytometer. Several multiplexed bead systems are currently marketed by different vendors. We have used the Luminex FlowMetrix system which consists of 64 different bead sets manufactured with uniform, distinct proportions of red and orange fluorescent dyes (detected by FL2/FL3 on a FACScan). Each bead set forms the basis of an individual assay using a green fluorescent reporter dye (FL1). This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. They can also be developed into rapid, 'no wash' assays that can be completed in <2 h. This review traces the historical association between microspheres and flow cytometry, the development and use of particle-based flow cytometric assays, how they compare with current assays and potential future developments of this very exciting technology.     

Rapid simultaneous measurement of multiple cytokines using 100 microl sample volumes--association with neonatal sepsis

Early diagnosis of neonatal infection has proved problematic due to the inadequacy of currently available laboratory tests. Neonatal sepsis is associated with an increase in plasma-derived cytokine levels, but an increase of a single cytokine cannot identify neonatal sepsis specifically and multiple cytokine levels are required. The time constraints and relatively large volume of plasma required to measure multiple cytokines from newborn infants by conventional enzyme-linked immunosorbent assay (ELISA) techniques is prohibitive. We therefore applied cytometric bead array (CBA) technology for simultaneous measurement of multiple cytokines from a group of 18 term neonates with infection confirmed by culture and a control group. 'Normal' ranges were established for each cytokine from 1-7-, 8-14- and 15-21-day-old newborns. There was no significant change in the levels of cytokines from infants in different control age groups, suggesting that basal cytokine levels are unchanged in the first 3 weeks of life. In the patient groups, however, there was a significant difference in several cytokines between the different age groups. Interleukin (IL)-6, IL-10 and IL-12 were increased significantly in the 1-7-day-old patient group compared to either the 8-14 and 15-21 age group, suggesting that infection in utero is associated with increased levels of these cytokines compared to infection acquired following birth. When individual patient cytokine levels were compared to normal control reference ranges, two patients failed to show significant elevation of any cytokine tested. All other patients showed elevated levels of between one and nine cytokines tested (mean of 4.6). There was no correlation between elevated cytokine levels and types of infective organism or patient age. In conclusion, neonatal sepsis is associated with the elevation of multiple plasma cytokines. The use of CBA kits is a rapid, easy, low sample volume and sensitive method to measure multiple plasma cytokines.     

Microspheres, nanospheres and flow cytometry: from cellular to molecular analysis

Micro- and nanospheres are tightly associated with the development of flow cytometry. They are indispensable tools to optimize diffraction and fluorescence signals as well as for fluorescence calibration and cellular purification (magnetic micro- and nanospheres). They are also usefull to evaluate phagocytosis and to detect slightly expressed antigens. Recently, developments of microspheres-based flow cytometric assays have raised to quantify soluble analytes in biological fluids, cellular and tissue samples. The technology utilizes spectrally distinct fluorescent microspheres as a solid support for a conventional immunoassay, affinity assay or DNA hybridisation assay which is subsequently analyzed on a flow cytometer. Several multiplexed bead systems are now available facilitating the development of multiplexed assays that simultaneously measure many different analytes in few microliters of sample. Some recent applications with fluorescent microspheres coated with antibodies or oligonucleotides include cytokines and PCR products quantitation and single nucleotide polymorphism genotyping. Thus, multiplex assays using microspheres and flow cytometry technologies are exciting techniques which have the potential to contribute to the development of efficient diagnostic and research methods.     

Development and validation of a multiplex microsphere-based assay for detection of domestic cat (Felis catus) cytokines

Cytokines are essential signaling molecules that mediate the innate immune response, and therefore their presence can be of diagnostic, prognostic, and pathogenic significance. Microsphere-based immunoassays allow rapid and accurate evaluation of cytokine levels in several species, including humans, dogs, and mice; however, technology to evaluate domestic cat (Felis catus) cytokines has been limited to single-analyte enzyme-linked immunosorbent assays (ELISAs). Microsphere-based immunoassays provide an attractive alternative technology for detecting and quantifying multiple analytes in a single assay using as little as 50 μl of sample. We describe the development and validation of a microsphere-based assay for three commonly analyzed domestic cat cytokines (gamma interferon, interleukin-10, and interleukin-12/interleukin-23 p40) using reagents from commercially available ELISAs. The assay was optimized for capture and detection antibody concentrations, streptavidin-phycoerythrin concentration, and number of microspheres. The validated lower and upper quantitation limits were 31 and 1,000 pg/ml for gamma interferon, 63 and 2,000 pg/ml for interleukin-10, and 39 and 625 pg/ml for interleukin-12/interleukin-23 p40. Cytokine concentrations in peripheral blood mononuclear cell supernatants were measured, and results obtained by the microsphere assay were correlated with values obtained with commercially available ELISA kits. This technology is a convenient and reproducible assay to evaluate domestic cat cytokine responses elicited by a variety of diseases.     

Evaluation of multiplex flow cytometric opsonophagocytic assays for determination of functional anticapsular antibodies to Streptococcus pneumoniae

The determination of functional antipneumococcal capsular polysaccharide antibodies by sequential testing of pre- and postvaccination serum samples one serotype at a time is sample-intensive and time-consuming and has a relatively low throughput. We tested several opsonophagocytic assay (OPA) formats, including the reference killing method, a monovalent bacterium-based flow method, a trivalent bacterium-based flow method, and a tetravalent bead-based flow method using a panel of sera (4 prevaccination and 16 postvaccination, from healthy adults immunized with the 23-valent pneumococcal polysaccharide vaccine). The trivalent and tetravalent methods allow simultaneous measurements of opsonic antibodies to multiple pneumococcal serotypes. The trivalent bacterial-flow OPA had significant correlation to the reference OPA method and to a previously published flow cytometric OPA (r values ranged from 0.61 to 0.91, P < 0.05) for serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. The tetravalent OPA had significant correlation to all OPA method formats tested (r values from 0.68 to 0.92, P < 0.05) for all seven serotypes tested. This tetravalent OPA is an alternative to other OPA methods for use during vaccine evaluation and clinical trials. Further, the flow cytometric multiplex OPA format has the potential for expansion beyond the current four serotypes to eight or more serotypes, which would further increase relative sample throughput while reducing reagent and sample volumes used.     

Binding of von Willebrand factor to collagen by flow cytometry

We developed a new method for the detection of large von Willebrand factor (vWf) multimers binding to collagen and for the determination of vWf antigen (vWf:Ag) using flow cytometry. Collagen is coated on to polystyrene beads, allowing detection of found large vWf multimers. In addition, rabbit antibody against vWf is coated on to the beads allowing detection of all vWf:Ag. In plasma samples from healthy persons and patients (with type 1, 2A, 2N, or severe von Willebrand disease or hemophilia), 4 different assays were performed: vWf:Ag by immunoelectrophoresis; vWf ristocetin cofactor (vWf:RCof); CBA; and vWf:Ag based on an enzyme-linked immunosorbent assay using polystyrene beads. We assayed the flow cytometric method using 2 bead sizes. The optimal bead size was 3.136 microns. The results of CBA and vWf:Ag closely correlated with those of vWf:RCof and vWf:Ag (immunoelectrophoresis), respectively, and showed a low limit of detection. Interassay variance of cytometric methods was lower than interassay variance of traditional assays. In addition, we used the new assays to monitor desmopressin therapy.     

A deep profiler's guide to cytometry

In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.     

A flow cytometric opsonophagocytic assay for measurement of functional antibodies elicited after vaccination with the 23-valent pneumococcal polysaccharide vaccine.

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5, 6-carboxyfluorescein, succinimidyl ester-labeled Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with >/=97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n = 28) and postvaccination (n = 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r = 0.89; P 相似文献   

Modeling and Optimization of High-Sensitivity,Low-Volume Microfluidic-Based Surface Immunoassays

Microfluidics are emerging as a promising technology for miniaturizing biological assays for applications in diagnostics and research in life sciences because they enable the parallel analysis of multiple analytes with economy of samples and in short time. We have previously developed microfluidic networks for surface immunoassays where antibodies that are immobilized on one wall of a microchannel capture analytes flowing in the microchannel. This technology is capable of detecting analytes with picomolar sensitivity and from sub-microliter volume of sample within 45 min. This paper presents the theoretical modeling of these immunoassays where a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We then derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance, which we think will allow further development of microfluidic networks for immunoassay applications.     

Evaluation of the new multiplexed immunoassay, FIDIS, for simultaneous quantitative determination of antinuclear antibodies and comparison with conventional methods

Our study was done to evaluate the FIDIS Connective kit (Biomedical Diagnostics, Marne la Vallée, France) for simultaneous quantitative determination in the same sample of 9 antinuclear antibody specificities directed against double-stranded DNA, SSA, SSB, Sm, Sm/RNP, Scl-70, Jo-1, ribosome, and centromere B and to compare it with standardized commercial methods, enzyme immunoassay and immunofluorescence. FIDIS technology constitutes a new multiplexed method using the Luminex 100 system (Luminex, Austin, TX) based on the use of distinct color-coded particles and flow cytometric detection. Serum samples from people with diagnosed rheumatic diseases with well-identified markers of autoimmunity were tested by a retrospective study. Specificity was assessed by testing blood donors and potential biologic interfering samples. This first evaluation demonstrated the analytic performance of FIDIS technology. Concordances with routine methods were between 99.1% and 100.0% on 222 samples. FIDIS was reliable (coefficients of variation < 10%) and accurate (correlation coefficients with enzyme-linked immunosorbent assay between 0.90 and 0.97) in a large measure range.     

Assessment of cellular activation by flow cytometric methods

The in vitro measurement of lymphoid cell activation is a valid correlate of immunological function in human subjects. This process can be evaluated by the assessment of the proliferative response or the cytokine synthesis and secretion by lymphoid cells upon their proper stimulation. The technology of flow cytometry provides unique conditions for the development of biological assays to achieve those purposes. In this work we describe the utilization of flow cytometric DNA analysis in the evaluation of: (1) the mitogenic response of lymphoid cells; (2) the cellular activity in mixed lymphocyte cultures; (3) the presence of interleukin-2 in culture supernatants, and (4) the cell cycle progression, simultaneous to the expression of a nuclear activation antigen among proliferating lymphocytes.     

Comparison of the GenMark Diagnostics eSensor Respiratory Viral Panel to Real-Time PCR for Detection of Respiratory Viruses in Children

A novel eSensor respiratory viral panel (eSensor RVP) multiplexed nucleic acid amplification test (GenMark Diagnostics, Inc., Carlsbad, CA) was compared to laboratory-developed real-time PCR assays for the detection of various respiratory viruses. A total of 250 frozen archived pediatric respiratory specimens previously characterized as either negative or positive for one or more viruses by real-time PCR were examined using the eSensor RVP. Overall agreement between the eSensor RVP and corresponding real-time PCR assays for shared analytes was 99.2% (kappa = 0.96 [95% confidence interval {CI}, 0.94 to 0.98]). The combined positive percent agreement was 95.4% (95% CI, 92.5 to 97.3); the negative percent agreement was 99.7% (95% CI, 99.4 to 99.8). The mean real-time PCR threshold cycle (C_T) value for specimens with discordant results was 39.73 (95% CI, 38.03 to 41.43). Detection of coinfections and correct identification of influenza A virus subtypes were comparable between methods. Of note, the eSensor RVP rhinovirus assay was found to be more sensitive and specific than the corresponding rhinovirus real-time PCR. In contrast, the eSensor RVP adenovirus B, C, and E assays demonstrated some cross-reactivity when tested against known adenovirus serotypes representing groups A through F. The eSensor RVP is robust and relatively easy to perform, it involves a unique biosensor technology for target detection, and its multiplexed design allows for efficient and simultaneous interrogation of a single specimen for multiple viruses. Potential drawbacks include a slower turnaround time and the need to manipulate amplified product during the protocol, increasing the possibility of contamination.     

A Flow Cytometric Opsonophagocytic Assay for Measurement of Functional Antibodies Elicited after Vaccination with the 23-Valent Pneumococcal Polysaccharide Vaccine

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5,6-carboxyfluorescein, succinimidyl ester-labeled Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with ≥97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n = 28) and postvaccination (n = 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r = 0.89; P ≤ 0.01) was found between the two assays for the seven serotypes tested. The flow cytometric assay is rapid (~4 h) with high throughput (~50 serum samples per day per technician) and provides a reproducible measurement of serotype-specific functional antibodies, making it a highly suitable assay for the evaluation of the immune responses elicited by pneumococcal vaccines.     

Flow cytometric microsphere-based immunoassay: analysis of secreted cytokines in whole-blood samples from asthmatics.

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-gamma than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.     

Sample preparation: the weak link in microfluidics-based biodetection

As a broad generalization, clinicians and laboratory personnel who use microfluidics-based automated or semi-automated instrumentation to perform biomedical assays on real-world samples are more pleased with the state of the assays than they are with the state of the front-end sample preparation. The end-to-end procedure requires one to collect, manipulate, prepare, and analyze the sample. The appeal of microfluidics for this procedure is partly based on its combination of small size and its ability to process very small liquid volumes, thus minimizing the use of possibly expensive reagents. However, real-world samples are often large and incompatible with the input port and the μm-scale channels of a microfluidic device, and very small liquid volumes can be inappropriate in analyzing low concentrations of target analytes. It can be a worthy challenge to take a raw sample, introduce it into a microfluidics-based system, and perform the sample preparation, which may include separation and concentration of the target analytes, so that one can benefit from the reagent-conserving small volumes and obtain the correct answer when finally implementing the assay of interest.

Multiplex measurement of proinflammatory cytokines in human serum: comparison of the Meso Scale Discovery electrochemiluminescence assay and the Cytometric Bead Array

Serum cytokine profiling is a powerful tool to link host immune defense with disease pathogenesis. Although several multiplex assays are commercially available, none has been rigorously validated in the context of chronic infectious disease (such as HIV infection). Here we compared the measurement of proinflammatory cytokines by two multiplex platforms: the Meso Scale Discovery (MSD) electrochemiluminescence assay and the Becton Dickinson Cytometric Bead Array (CBA) flow cytometric assay, using serum samples from HIV-infected and -uninfected donors. We evaluated the ability of these assays to: a) quantify circulating levels of native cytokines (IL-6, IL-8, IL-10, TNF-α, IL-12p70, IL-1β), and b) accurately recover known amounts of recombinant cytokines added to serum samples. Based on the standard curves, the sensitivity of the MSD system was only slightly better than the CBA. However, in serum the MSD platform consistently quantified levels of endogenous IL-12p70, TNF-α, and IL-10 that were undetectable by the CBA assay. The MSD assay was also more accurate as determined by an enhanced capacity to recover known concentrations of recombinant cytokines added to serum. Both assays performed equally well in quantifying IL-6 and IL-8, while neither assay quantified IL-1β with accuracy and precision. Interestingly, HIV infection did not affect the performance of either assay. Overall, the MSD assay provided a more reliable assessment of the proinflammatory cytokines tested in the serum of healthy and HIV-infected individuals.     

Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-γ (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-γ than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.     

Immunoaffinity solid-phase extraction for the trace-analysis of low-molecular-mass analytes in complex sample matrices

Immunoaffinity solid-phase extraction (SPE) sorbents, so-called immunosorbents (ISs), are based upon molecular recognition using antibodies. Thanks to the high affinity and high selectivity of the antigen-antibody interaction, they allow a high degree of molecular selectivity and have shown to be a unique tool in the sample preparation area these last few years. Extraction and clean-up of complex biological and environmental aqueous samples are achieved in the same step and from large volumes when required. Their application to extracts from solid matrixes is solvent-free and more simple than any other clean-up procedure. Single analytes can be targeted, but since an antibody can also bind one or more analytes having structure similar to the one used for its preparation, ISs have been developed for targeting a single analyte and its metabolites. The cross-reactivity was also exploited for developing ISs that could selectively extract a whole class of structurally related compounds. This review describes the current technology used for the synthesis of the ISs, their properties and their field of application. The different parameters governing the antigen-antibody interactions and the solid-phase extraction process are discussed. Emphasis is given to the optimisation of the SPE sequence, especially to the desorption and regeneration steps. The importance of the capacity and its relationship with the analytes recovery and breakthrough volumes is highlighted for class-specific ISs. Multi-class-selective ISs are also presented. Validation studies are reviewed using various certified reference materials. Relevant examples, involving combination with chromatography in both off-line and on-line mode, illustrate the high selectivity provided in various complex matrixes. Miniaturisation is also described, since it allows high throughput of samples.