Analysis of T lymphocyte reactivity to complex antigen mixtures by the use of proteins coupled to latex beads

This report describes a suitable model for analysing heterogeneous T cell responses to complex foreign antigens using coated polystyrene beads. The advantage of this technique is that it allows the simple removal of detergents from bound antigen so that biochemically separated antigens or crude antigen mixtures can be used. Furthermore, due to the enhanced uptake of latex-bound antigens by phagocytic antigen-presenting cells (APC), very small amounts of antigen will suffice for activation of T cells in vitro. The potential use of this technique to analyse relevant T cell responses to antigens which are difficult to obtain purified in bulk quantities, is discussed.     

Microbeads,nanobeads and cytometry: applications to the analysis and purification of cells and biomolecules

Nano and microspheres are important tools in cytometry. They have been used in first to optimize fluorescent signals detected by flow cytometry and to evaluate phagocytosis. Some antigens were also detected by using nanospheres covalently coupled to antibodies. Specifically dedicated microspheres are now widely used for antigenic quantitation by flow cytometry, and magnetic nano and micropheres are very usefull for cellular and molecular purifications. To date, analytical methods based on the use of microspheres are developed to detect proteins, nucleic acids, and ions. To this end, antibodies, oligonucleotides, or chelating agents are bound to microspheres characterized by different fluorescences. The applications of these multiplexed microspheres assays allow to identify and quantify simultaneously some macromolecules and ions, but they also permit to analyze enzymatic activities and to perform polymorphism analyses. With microspheres used as reactive support, molecular analyses are therefore possible by flow cytometry. Nano and microspheres are also usefull tools for calibration in confocal microscopy as well as for micromanipulations of biomolecules and of living cells. Inovative methods based on the use of nano and microspheres are expected in the fields of biology, medicine, food industry, and environmental sciences.     

A Rapid Microplate-Based Fluorometric Assay for Phagocytosis

A highly sensitive quantitative fluorometric assay for phagocytosis, previously measured using fluorescence spectrophotometry or flow cytometry, has been adapted for use with a 96-well fluorescence plate reader. The technique allows rapid analysis of large numbers of samples, and requires only a small sample volume. Comparison of plate types demonstrated that opaque white 96-well luminostrips produced a 100 fold greater fluorescent output, and were more sensitive than black fluoroplates. Intraplate variability was also significantly lower using white luminostrips. For the phagocytic assay, fluorescein conjugated polystyrene beads were incubated with macrophage monolayers in white luminostrips. After incubation, cells were washed, lysed and phagocytosis quantified by determining the fluorescent intensity using a fluorescence plate reader. The number of beads phagocytized was determined from a standard curve of bead number versus fluorescent output. The phagocytic activity of resident and thioglycollate-elicited peritoneal macrophages was compared using this technique.     

A Rapid Microplate-Based Fluorometric Assay for Phagocytosis

A highly sensitive quantitative fluorometric assay for phagocytosis, previously measured using fluorescence spectrophotometry or flow cytometry, has been adapted for use with a 96-well fluorescence plate reader. The technique allows rapid analysis of large numbers of samples, and requires only a small sample volume. Comparison of plate types demonstrated that opaque white 96-well luminostrips produced a 100 fold greater fluorescent output, and were more sensitive than black fluoroplates. Intraplate variability was also significantly lower using white luminostrips. For the phagocytic assay, fluorescein conjugated polystyrene beads were incubated with macrophage monolayers in white luminostrips. After incubation, cells were washed, lysed and phagocytosis quantified by determining the fluorescent intensity using a fluorescence plate reader. The number of beads phagocytized was determined from a standard curve of bead number versus fluorescent output. The phagocytic activity of resident and thioglycollate-elicited peritoneal macrophages was compared using this technique.     

An immunofluorescence reaction for Schistosoma mansoni using the defined antigen substrate spheres (DASS) system

Schistosoma mansoni antigens covalently bound to agarose beads were found suitable as a matrix for an immunofluorescent reaction on S. mansoni with sera of infected animals and men. This system proved to be at least as sensitive as the indirect fluorescent antibody reaction with frozen sections of the adult parasite; furthermore, storage of the antigen-bound beads in freeze-dried state proved to be possible. Automated microfluorometry permitted the rapid measurement of a sufficient number of beads for routine applications. The DASS-system is a promising quantitative method for the identification of a variety of antigens using the indirect immunofluorescence method.     

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 micron 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.     

巨细胞病毒pp65抗原检测的新方法

目的传统的检测CMVpp65抗原的方法是间接免疫荧光,操作麻烦,步骤较多,整个实验需要6个多小时,而且只能定性。需要寻找一种简便的可以定量的方法来检测CMVpp65抗原阳性细胞。因此我们设计了流式的方法来检测CMVpp65抗原阳性细胞。方法我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行了流式标记移植病人外周血中的WBC,操作步骤明显简便了,只需不到2 h就可完成检测,而且可以定量检测CMVpp65抗原阳性细胞。同时我们用荧光定量PCR检测病人尿中的CMV基因的拷贝数。结果流式检测阳性率高的标本,用间接免疫荧光方法检测也为阳性,同时流式检测强阳性的病人尿用荧光定量PCR检测到CMV基因,表明病人处于CMV病毒血症。结论我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行流式标记,成功检测了移植病人的CMVpp65抗原阳性细胞的百分比,新方法可以定量,与荧光定量PCR方法检测病人尿中的CMV基因的结果符合率较高,可以推广应用。     

Quantitative flow cytometry in the clinical laboratory

Flow cytometry is most often used in the clinical laboratory for the purpose of immunophenotyping. Here, fluorescently labeled antibodies are bound to cell surface receptors, and their presence on the cell is most often defined in bivariate terms of positive or negative, with a cutoff set relative to a nonstaining control population. It has long been recognized that the intensity of the fluorescent signal is proportional to the amount of antibody bound per cell and therefore related to the number of antigen sites expressed. This relationship makes flow cytometers, at least theoretically, capable of quantifying antigen expression in terms of molecules per cell. There were numerous obstacles to the development of such methods and clinical utilization of fluorescence intensity measures by flow cytometry has in the past been largely overlooked. The first widespread recognition of the clinical utility for fluorescence intensity measures came from laboratories where malignant phenotypes were defined by aberrant intensity of staining due to over or under expression of various cellular proteins. These semiquantitative measures were relative in nature and described staining as bright or dim compared to that normally seen in healthy individuals. Recent advances within the past decade have resulted in the development of flow cytometric methods and materials that now permit one to conduct measures of quantitative fluorescence with improved levels of control and interlaboratory precision. With these advances have come increasing interest in quantitative flow cytometry as a method to quantify the expression and activities of a variety of proteins and enzymes for diagnostic, prognostic, and therapeutic purposes. This article discusses the background and theoretical and practical considerations, as well as the current use of quantitative flow cytometry measures in the clinical laboratory.     

Demonstration of cytoplasmic and nuclear antigens in acute leukaemia using flow cytometry.

AIMS--To detect cytoplasmic and nuclear antigens using flow cytometry in acute leukaemia and to use this technique for double marker combinations. METHODS--Cytoplasmic staining was carried out in samples from 40 cases of acute leukaemia with monoclonal antibodies against the myeloid antigen CD13, the lymphoid antigens CD3, CD22, mu chain and the enzymes terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO). The cells were fixed with paraformaldehyde and permeabilised with Tween 20 and Becton Dickinson's FACS lysing solution. Flow cytometry results were compared in the same cases with immunocytochemistry results using the alkaline phosphatase anti-alkaline phosphatase method. RESULTS--The gentle permeabilisation induced by this method permitted preservation of the membrane antigens and the size and morphology of the cells. The results using flow cytometry were comparable with those obtained using immunocytochemistry, with nearly complete concordance in most cases. CONCLUSIONS--This technique is simple, rapid, sensitive and reproducible and it is suitable for double staining procedures, such as nuclear and cytoplasmic, nuclear and membrane, or cytoplasmic and membrane. It therefore provides a powerful tool for extending the use of immunophenotyping for the diagnosis and follow up of acute leukaemia. It could also be used for the investigation of minimal residual disease.     

Microfluorometry on antigen-antibody interaction in immunofluorescence using antigens covalently bound to agarose beads

Antigens covalently bound to agarose beads were found to be suitable for microfluorometry on individual beads of fluorescein-conjugated antibodies reacting with these antigens. With this technique, the interaction of conjugates with IgG, IgM and ovalbumin bound to the beads was studied. The system was found to be applicable to specificity tests. It also appeared to be more sensitive for high conjugate dilutions than immunofluorescence methods using tissue sections or cells of defined antigenicity as a substrate.     

Rapid method for detection of influenza a and B virus antigens by use of a two-photon excitation assay technique and dry-chemistry reagents

New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.     

Quantitative two-photon flow cytometry--in vitro and in vivo

Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.     

Serodiagnostic application of immunohistoperoxidase reactions on antigen-coupled agarose beads.

Agarose beads to which antigens were covalently bound were subjected to indirect immunohistoperoxidase procedures. For detection and titration of serum antibodies against bovine gamma globulin (BGG) this method appeared to be specific and sensitive. One advantage is that no special instruments are needed. As an example of diagnostic applicability the system was successfully used for demonstration of antibodies in human serum containing antibodies against the trematode Schistosoma mansoni. At the microscopical level the technique is suitable for study of basic problems. Microspectrophotometric absorbance scanning of the beads revealed that the method can provide quantitative information, and is probably capable of quantifying stoichiometric relations in immunological reactions.     

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.     

B cells do not present antigen covalently linked to microspheres.

B cells have been shown to present antigen to T cells very efficiently through their capacity to capture antigens by their membrane immunoglobulin. This direct cognate interaction of T and B cells results in the proliferation and differentiation of B cells. This concept has been established using soluble proteins. However, most of the antigens to which the immune system is exposed are included in complex particulate structures such as bacteria or parasites. The capacity of B cells to present these large and complex antigens is still unclear. To address this question we have studied the presentation by trinitrophenyl (TNP)-specific B cells of the same antigen TNP-KLH (keyhole limpet haemocyanin), either in a soluble form or covalently linked to poly(acrolein) microspheres, from 0.25 to 1.5 microns in diameter. In the presence of irradiated splenocytes or purified macrophages as a source of antigen-presenting cells (APC), KLH-specific T cells proliferated in response to soluble TNP-KLH or to TNP-KLH coupled to beads. In contrast, TNP-specific memory B cells were totally ineffective in presenting the TNP-KLH beads to KLH-specific T cells whereas they presented very efficiently soluble TNP-KLH. Similar results were obtained with the A20 B lymphoma or with lipopolysaccharide (LPS)-activated TNP-specific B cells. These results therefore indicate that B cells are unable to present large size particulate antigens such as bacteria or parasites.     

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.     

Flow cytometry in immunohematology]

Flow cytometry is an objective, sensitive and quantitative technique which allows rapid and simultaneous analysis of several parameters on a great number of cells. Hence, flow cytometry is particularly suitable for the analysis of complex cell populations, rare events and quantitative studies. In immunohematology, flow cytometry is a very powerful approach to the study of mixed red cell populations (hematopoietic chimerism, transfusion or bone marrow transplantation), the detection of low frequency cell populations (reticulocytes, fetomaternal hemorrhage) and the quantitative analysis of red blood cell antigens.     

Glomerular filtration rate and microsphere distributions in single nephron of rat kidney

In control non diuretic (ND) and in salt-loaded (SL) rats, both the microsphere technique and the 14C ferrocyanide infusion technique were used to determine the distribution of microspheres in single glomeruli and the SNGFR of the corresponding nephrons. A sample of microspheres with a diameter averaging 11.0 +/- 2.6 mu SD was selected from a 15 +/- 5 mu unlabelled batch. In each rat, three million of these microspheres were injected through the left carotid artery. The microspheres were directly counted under microscopic observation in the glomeruli of the nephrons which were microdissected to determine the SNGFR value. The number of microspheres per glomerulus for a given kidney generally varied from 0 to 8 and was independent to the SNGFR value. The diameter of the microspheres trapped was constant in all the animals. The mean number of microspheres for superificial (S) and juxtamedullary (JM) nephrons was, (ND); S = 1.99 +/- 0.48 SE., n = 5; JM = 3.02 +/- 0.51 SE, n = 5; P less than 0.02, (SL): S = 3.75 +/- 0.53 SE, n = 6; JM = 2.86 +/- 0.33 SE, n = 6; P less than 0.05. This distribution was directly related to that of SNGFR in ND rats (S = 39.0 +/- 6.1 SD and JM = 49.5 +/- 10.3 nl/min) but not in SL rats (S = 50.9 +/- 6.1 and JM = 66.9 +/- 10.0 nl/min). In conclusion, the microsphere technique described in the present paper, appears more suitable for investigating single glomerular blood flow since the number and the size of the microspheres trapped are directly determined at the level of the glomerulus.     

Extracorporeal removal of specific antibodies by hemoperfusion through the immunosorbent agarose-polyacrolein microsphere beads: removal of anti-bovine serum albumin in animals

We describe the production of a unique immunosorbent system, agarose-polyacrolein microsphere beads (APAMB) for removal of a specific antibody, anti BSA, and its efficacy in animal trials. This is a model system for hemoperfusive removal of specific antibodies or antigens directly from whole blood. The agarose beads (1.0 mm mean diameter) contain thousands of microspheres of 0.2 micron mean diameter. The microspheres which contain the ligand are encapsulated within an agarose matrix to confer physical strength, biocompatibility, spacial configuration, and porosity allowing rapid entry of plasma for reaction. Any antigen may be linked covalently to spacers on the polyacrolein microspheres to remove a specific antibody, or vice versa. Thus the APAMB remove specific molecules in contrast to the charcoal or ion exchange resins currently in use. Removal of antibody is efficient and rapid, therefore, short hemoperfusive times may be used. The beads are biocompatible; there are negligible decreases in RBC, WBC and platelets. Electrolytes and other soluble components also are minimally affected. Therapy, at the least palliative, of autoimmune disorders i.e., multiple myeloma, macroglobinemia, autohemolytic anemias, idiopathic thrombocytopenia, myasthenia gravis, rheumatoid arthritis, thyroiditis, glomerulonephritis, etc, is potentially available with this or its further improved versions.     

Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.