Microsphere-based Immunoassay Research Articles

Multiplexed microsphere-based flow cytometric immunoassays for human cytokines

Cytokines play a pivotal role in the regulation of immunologic, hematologic and wound-healing processes. They function to stimulate as well as inhibit the proliferation, differentiation and maturation of a variety of cell types. Thus, their functions are pleiotropic as well as interdependent to the extent that any cytokine may have effects that are synergistic or antagonistic with other cytokines. Cytokines also display redundancy when one mimics the functions of others. These characteristics imply that measuring the levels of one cytokine in a biologic system provides only a fraction of the information that is relevant to the existing physiologic state. A more realistic indication of the complexity of cellular interactions would include measurements of multiple cytokines at any time point. One method of multiplexed analysis can be performed by capture of the cytokines on an array of fluorescent microspheres for quantitation by flow cytometry. This technology has been applied to a variety of biomolecules, but simultaneous quantitation of multiple cytokines in a small sample volume has become rapid, inexpensive, reliable and informative.

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.

Simultaneous measurement of antibodies to three HIV-1 antigens in newborn dried blood-spot specimens using a multiplexed microsphere-based immunoassay.

We developed a fluorescent immunoassay to simultaneously measure antibodies to three HIV-1 antigens from newborn dried blood-spot specimens. The multiplexed assay uses fluorescent microspheres and a flow analyzer. The procedure is sensitive, precise and accurate, and can be expanded to simultaneously measure additional multiple analytes from a single specimen.

A Duplexed Microsphere-Based Fluorescent Immunoassay

Microsphere-based immunoassays are described for the simultaneous measurement of the clinically important drugs digoxin and theophylline. Competitive immunoassays were performed using haptenized microspheres and antibodies labeled with horseradish peroxidase. Enzyme-catalyzed reporter deposition (CARD) resulted in immunofluorescence signal amplification. Two encoding dyes were used to differentiate analytical signals from microspheres containing assays for the two analytes. An epifluorescence microscope and a CCD camera interfaced with a computer were utilized to measure fluorescence signals of individual microspheres. The microspheres from a duplexed assay were mounted on microscope slides as well as inserted into wells etched into the distal ends of optical imaging fibers. Fluorescence images from both formats were captured. In the experiments using microscope slides, the immunoassays were successfully duplexed and only marginal interferences at high analyte concentrations were observed. Preliminary results suggest that simultaneous determination of the two analytes using a fiber-based sensor-array format is feasible, but requires further development before precise quantitative analyses are possible.

Platelet adhesion onto artificial surfaces: Inhibition by benzamidine, pentamidine, and pyridoxal-5-phosphate as demonstrated by flow cytometric quantification of platelet adhesion to microspheres

An appreciable effort is directed toward designing strategies to minimize platelet interactions with artificial surfaces, because their reactivity is thought to promote thrombus formation and lead to materials failure. Although platelet glycoprotein Ib/IX (GPIb/IX) and glycoprotein IIb/IIa (GPIIb/IIIa) receptors are thought to mediate adhesion, whether GPIIb/IIIa receptors are activated and how this might occur are largely unknown and are the focus of this article. There are a few ways, other than thrombin generation, that blood contact with artificial surfaces can lead to GPIIb/IIIa activation. Complement activation can lead to products capable of activating platelets (C1q, C5b-9), and contact between platelet CD32 (FcγRII) receptors and immobilized immunoglobulin G could also activate platelets. In this article the potential role of these processes was evaluated by using various inhibitors in a microsphere-based platelet adhesion immunoassay. Polystyrene microspheres (10 μm) were incubated in platelet-rich plasma before flow cytometric analysis of beads for adherent platelets. The data eliminated occupancy of the FcγRll receptor (by use of IV.3 blocking antibody), C5b-9 production (by use of sCR1), and the indirect action of factor XIIa on complement components (by use of corn trypsin inhibitor) as playing roles in supporting platelet adhesion. Agents directed against the first complement component (benzamidine, pentamidine, pyridoxal-5-phosphate) were effective inhibitors of platelet adhesion and were also demonstrated to inhibit SC5b-9 and C3d levels on the bead surface after serum incubations. Because these agents are not highly specific, it can not be concluded that C1q is a mediator of adhesion. These agents were also demonstrated to inhibit fluorescein isothiocyanate-fibrinogen binding to activated washed platelets, therefore indicating that fibrinogen receptor expression is a requirement for platelet adhesion.

The sensitive detection and quantitation of antibody to HVC by using a microsphere-based immunoassay and flow cytometry

Antibody to HCV core and NS3 was quantified by using a microsphere immunoassay and flow cytometry. Antibody to core and NS3 was elevated in the 85 seropositive blood donors tested. The amount of either antibody varied over two logs although greater variation was seen with the antibody to NS3 than was seen with antibody to core. In three documented acute HCV cases, the microsphere assay detected antibody prior to antibody detection using the reference methods. Twenty donor samples were indeterminate by the reference methods: 45% of these were indeterminate with the microsphere assay while 25% were negative and 30% were positive. As compared to enzyme immunoassay the microsphere assay showed a 5-fold increase in sensitivity. The microsphere assay demonstrated increased sensitivity for the quantification of specific antibody to HCV core and NS3 and was useful in resolving a significant proportion of indeterminate samples.

Development of a microsphere-based fluorescent immunoassay and its comparison to an enzyme immunoassay for the detection of antibodies to three antigen preparations from Candida albicans

A sensitive assay for the simultaneous detection of multiple serum antibodies by flow cytometry was developed. Polystyrene microspheres of 5, 7 and 9.3 μm in diameter were used as solid supports for the attachment of three different antigen preparations from Candida albicans. These antigens were a whole cell extract, a cytoplasmic protein extract and a cell wall polysaccharide. Microsphere-associated fluorescence was quantitated by flow cytometry, with the different sized microspheres analyzed separately using electronic volume gating. This procedure allowed for different antigen-coated microspheres with discrete sizes to be analyzed independently for immunofluorescence. The assay detected antibody levels in human serum at dilutions up to 10 −6 and provided complete discrimination, using all three antigen preparations, between antibody levels seen in healthy subjects and those seen in patients suspected of having a systemic Candida infection. A standard enzyme immunoassay (EIA) failed to provide complete discrimination between healthy subjects and patient samples: at least 17% of patient values fell within the healthy subject range using all three antigen preparations. The microsphere assay which allowed for the simultaneous detection of multiple antibodies, has increased dynamic range over EIA and provides for better discrimination of patients from healthy subjects in comparison to EIA. Precise quantitation of antibodies is possible and the rapid analysis of thousands of microspheres markedly enhances the statistical accuracy of the assay. We suggest this assay is likely to have many other important applications in immunologic testing.

Simultaneous detection of antibodies to cytomegalovirus and herpes simplex virus by using flow cytometry and a microsphere-based fluorescence immunoassay.

A sensitive assay for the simultaneous detection of anti-cytomegalovirus and anti-herpes simplex virus antibodies was developed. Two different sizes of polystyrene microspheres were coated with purified viral antigens. Human antiviral antibodies were detected with a biotin-streptavidin amplification procedure with phycoerythrin as the fluorescent label. Microsphere-associated fluorescence was quantitated with a flow cytometer. Sixteen percent of samples initially scored as seronegative for cytomegalovirus and 35% of samples initially scored as seronegative for herpes simplex virus by conventional assays were clearly found positive by the microsphere technique. This flow cytometric assay can simultaneously detect several specific antibodies at levels which are below the sensitivity of standard assays. The dynamic range of this assay is at least sixfold greater than that of enzyme immunoassays. This technique is amenable to numerous serologic assays and could greatly expand the clinical laboratory applications of flow cytometry.