A Latex Bead-Based Flow Cytometric Immunoassay Capable of Simultaneous Typing of Multiple Pneumococcal Serotypes (Multibead Assay)

ABSTRACT A simple and rapid method of simultaneously determining 15Streptococcus pneumoniae serotypes was developed. Fifteen latex beads of different sizes and different red fluorescence levels were coated with 1 of 15 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, and 23F) of pneumococcal capsular polysaccharide (PS). The bead mixture was incubated with individual pneumococcal lysate, a pool of rabbit antisera capable of binding the 15 serotypes, and fluorescein (green fluorescence)-conjugated anti-rabbit antibody. Bead size, red fluorescence, and green fluorescence were measured in a single flow cytometer run. The green fluorescence of the beads was inhibited only when there was a serotypic match between PS on the bead and PS in the pneumococcal lysate. This method distinguished cross-reactive serotypes and correctly identified the serotypes in 100% of 86 pneumococcal isolates tested.

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Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions

Blood ◽

10.1182/blood.v63.4.768.768 ◽

1984 ◽

Vol 63 (4) ◽

pp. 768-778 ◽

Cited By ~ 73

Author(s):

CW Jackson ◽

LK Brown ◽

BC Somerville ◽

SA Lyles ◽

AT Look

Keyword(s):

Rapid Determination ◽

Marrow Cell ◽

Green Fluorescence ◽

Color Flow ◽

Red Fluorescence ◽

Flow Cytometric ◽

Clinical Investigations ◽

Flow Cytometric Measurement ◽

Fcm Analysis ◽

Cellular Dna

Abstract The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two- color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.

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Streptococcus pneumoniae Capsular Serotype 19F Is More Resistant to C3 Deposition and Less Sensitive to Opsonophagocytosis than Serotype 6B

Infection and Immunity ◽

10.1128/iai.01186-08 ◽

2008 ◽

Vol 77 (2) ◽

pp. 676-684 ◽

Cited By ~ 47

Author(s):

Merit Melin ◽

Hanna Jarva ◽

Lotta Siira ◽

Seppo Meri ◽

Helena Käyhty ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Conjugate Vaccine ◽

Middle Ear ◽

Complement Activation ◽

Capsular Polysaccharide ◽

Immune Defense ◽

Pneumococcal Serotypes ◽

Vaccine Trials ◽

Opsonophagocytic Killing ◽

Complement Deposition

ABSTRACT The polysaccharide capsule is a major virulence mechanism of Streptococcus pneumoniae, shielding the bacterium from phagocytes. Capsule types may differ in their abilities to resist immune defense. Antibody-mediated complement activation and opsonophagocytosis are crucial in protection against pneumococcus. Conjugate vaccine trials suggest imperfect protection against 19F. We have previously shown that significantly more anti-19F than anti-6B antibody is needed for killing in the opsonophagocytic assay (OPA). In this study, we explored whether the amount of C3 deposited on serotype 6B and 19F pneumococcal strains reflects their sensitivity to opsonophagocytosis. We compared clinical 6B and 19F nasopharyngeal, middle ear, and blood isolates as well as reference OPA strains (n = 16) for their sensitivity to opsonophagocytosis and C3 deposition. Sixfold anticapsular antibody concentrations were required for 50% opsonophagocytic killing of 19F compared to that of 6B strains. Serotype 19F was more resistant to C3 deposition than 6B. Complement deposition and opsonophagocytosis were dependent on the concentration of anticapsular antibodies. Differences between pneumococcal serotypes in antibody-mediated protection may partly be explained by the abilities of the capsules to resist complement deposition. These findings support previous studies suggesting that higher antibody concentrations to the capsular polysaccharide are needed for protection against disease caused by serotype 19F than that caused by 6B.

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Carbon Source-Dependent Changes of the Structure of Streptococcus pneumoniae Capsular Polysaccharide with Serotype 6F

International Journal of Molecular Sciences ◽

10.3390/ijms22094580 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4580

Author(s):

Joel P. Werren ◽

Lukas J. Troxler ◽

Oluwaseun Rume-Abiola Oyewole ◽

Alban Ramette ◽

Silvio D. Brugger ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Carbon Source ◽

1H Nmr ◽

31P Nmr ◽

Capsular Polysaccharide ◽

Defined Medium ◽

Flow Cytometric ◽

Unequivocal Identification ◽

Pneumococcal Serotype ◽

Polysaccharide Composition

The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

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Immunobiological Properties of Antigenic Preparations Streptococcus pneumoniae and their Mixtures

Epidemiology and Vaccinal Prevention ◽

10.31631/2073-3046-2021-20-6-5-11 ◽

2022 ◽

Vol 20 (6) ◽

pp. 5-11

Author(s):

M. M. Tokarskaya ◽

E. A. Nayаnova ◽

O. V. Nechaeva ◽

S. A. Baranovskaya ◽

O. M. Afanacyeva ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Capsular Polysaccharide ◽

Solid Phase ◽

Control Group ◽

Global Public Health ◽

Pneumococcal Serotypes ◽

Specific Antibodies ◽

Humoral Immune ◽

Apparent Decrease ◽

Stimulation Of

Relevance. Type-specific immunity does not protect against infection with other pneumococcal serotypes. The phenomenon of the change of serotypes dominating the population of Streptococcus pneumoniae is known, in part due to the intensive recombination process and the phenomenon of «capsule switching». Therefore, the development of a serotype-independent pneumococcal vaccine is an important global public health priority. Ams. Investigation of immunobiological properties of candidate components of a future vaccine with serotype-independent activity. Materials and methods. For immunization of mice, preparations of the capsular polysaccharide of pneumococcus serotype 3 (CPS) were used; protein-containing fraction (PCF) obtained from an aqueous extract of S. pneumoniae 6B cells; recombinant pneumolysin (Ply); mixtures of drugs (CPS + Plу; CPS + PCF; PCF + Plу); conjugate vaccine Prevnar 13 (manufactured by PFIZER Inc. USA). Mice were immunized intraperitoneally, 2 times with an interval of 14 days. Intact mice were used as a control group. To assess the humoral immune IgG response, the method of solid-phase ELISA was used. Phagocytic activity was studied at 7, 14, 21 and 28 days after the second immunization. The cytokine level was determined in the blood sera of mice after the second immunization 2, 4, 8, and 24 hours later on a NovoCyte flow cytometer (ACEA Biosciences, USA) using the MACSPIex CytoKine 10 Kit mouse (Miltenyi Biotec Inc., USA) according to the manufacturer's instructions. Results. Immunization of mice with Ply as well as mixtures with CPS and PCF caused a significant increase in the level of antibodies to Ply. It was found that there was no apparent decrease in the level of antigen-specific antibodies when antigens were administered in combination with others. Pneumolysin, used alone or in combination with PCF and CPS, induces the production of antiinflammatory cytokines IL-4, IL-10, and IL-5 detected throughout the study. This is confirmed by a study of the opsonophagocytic activity of neutrophils from immunized CPS + Ply, Ply + PCF and Ply mice; a significant increase in the number of eosinophils is observed in their blood due to the stimulation of their production of IL-5. Conclusions. As a result of the studies, it was shown that Ply, used alone or in combination with CPS and PCF, has the highest immunogenicity: it stimulates a significant increase in the level of specific antibodies, stimulates Th-2, and induces the production of anti-inflammatory cytokines.

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Two-color flow-cytometric analysis of the growth cycle of Plasmodium falciparum in vitro: identification of cell cycle compartments.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.12.2431031 ◽

1986 ◽

Vol 34 (12) ◽

pp. 1651-1658 ◽

Cited By ~ 19

Author(s):

J D Hare

Keyword(s):

Cell Cycle ◽

Plasmodium Falciparum ◽

Dna Synthesis ◽

Flow Cytometric Analysis ◽

Growth Cycle ◽

Green Fluorescence ◽

Red Fluorescence ◽

Color Display ◽

Flow Cytometric

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.

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Development of an Automated and Multiplexed Serotyping Assay for Streptococcus pneumoniae

Clinical and Vaccine Immunology ◽

10.1128/cvi.05312-11 ◽

2011 ◽

Vol 18 (11) ◽

pp. 1900-1907 ◽

Cited By ~ 40

Author(s):

Jigui Yu ◽

Jisheng Lin ◽

Kyung-Hyo Kim ◽

William H. Benjamin ◽

Moon H. Nahm

Keyword(s):

Streptococcus Pneumoniae ◽

Assay System ◽

Pneumococcal Vaccines ◽

Pneumococcal Serotypes ◽

Content Type ◽

Technical Challenge ◽

Multiplex Assays ◽

Flow Cytometric ◽

Array Technology ◽

Capsule Locus

ABSTRACTStreptococcus pneumoniaeexpresses more than 90 capsule types, and currently available pneumococcal vaccines are designed to provide serotype-specific protection. Consequently, serotyping of pneumococcal isolates is important for determining the serotypes to be included in pneumococcal vaccines and to monitor their efficacy. Yet serotyping of pneumococcal isolates has remained a significant technical challenge. By multiplexing many assays, we have now developed a simple yet comprehensive serotyping assay system that can not only identify all known pneumococcal serotypes but also subdivide nontypeable (NT) isolates into those with or without the conventional capsule locus. We have developed this assay system to require only six key reagents: two are used in one multiplex inhibition-type immunoassay, and four are required in two multiplex PCR-based assays. The assay system is largely automated by a seamless combination of monoclonal antibody-based and PCR-based multiplex assays using the flow cytometric bead array technology from Luminex. The assay system has been validated with a panel of pneumococci expressing all known pneumococcal serotypes and was found to be easily transferable to another laboratory.

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Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions

Blood ◽

10.1182/blood.v63.4.768.bloodjournal634768 ◽

1984 ◽

Vol 63 (4) ◽

pp. 768-778 ◽

Cited By ~ 9

Author(s):

CW Jackson ◽

LK Brown ◽

BC Somerville ◽

SA Lyles ◽

AT Look

Keyword(s):

Rapid Determination ◽

Marrow Cell ◽

Green Fluorescence ◽

Color Flow ◽

Red Fluorescence ◽

Flow Cytometric ◽

Clinical Investigations ◽

Flow Cytometric Measurement ◽

Fcm Analysis ◽

Cellular Dna

The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two- color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.

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Evaluation of Multiplex Flow Cytometric Opsonophagocytic Assays for Determination of Functional Anticapsular Antibodies to Streptococcus pneumoniae

Clinical and Vaccine Immunology ◽

10.1128/cvi.13.4.459-466.2006 ◽

2006 ◽

Vol 13 (4) ◽

pp. 459-466 ◽

Cited By ~ 20

Author(s):

Joseph E. Martinez ◽

Elizabeth A. Clutterbuck ◽

Han Li ◽

Sandra Romero-Steiner ◽

George M. Carlone

Keyword(s):

Capsular Polysaccharide ◽

Polysaccharide Vaccine ◽

Sequential Testing ◽

Pneumococcal Polysaccharide Vaccine ◽

Pneumococcal Serotypes ◽

Serum Samples ◽

Flow Method ◽

Flow Cytometric ◽

Sample Throughput

ABSTRACT 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.

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