Monitoring the insecticidal toxins fromBacillus thuringiensisin soil with flow cytometry

1997 ◽  
Vol 43 (11) ◽  
pp. 1074-1078 ◽  
Author(s):  
H. Tapp ◽  
G. Stotzky
Keyword(s):  
Flow Cytometry ◽  
Bacillus Thuringiensis ◽  
Internal Control ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Significant Shift ◽  
Dot Blot ◽  
Natural Habitats ◽  
Insecticidal Toxins ◽  
The Right

The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in environmental hazards, such as toxicity to nontarget species and the selection of toxin-resistant target species. We describe the use of flow cytometry as a method for detecting and tracking the fate of these insecticidal toxins in soil that does not require their extraction and purification. The toxins from B. thuringiensis subspp. tenebrionis and kurstaki were bound on clay- or silt-sized particles separated from Kitchawan soil that was unamended (naturally contains predominantly kaolinite) or amended to 6% v/v with the clay minerals montmorillonite or kaolinite (as an internal control). The particle–toxin mixtures were suspended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powder to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, and then resuspended in a solution of anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC–Ab). Controls consisted of the particles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstaki. All particles that bound the toxin from B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with the nonspecific fluorescence from the control FITC–Ab complexes with particles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity between the toxins from B. thuringiensis subspp. tenebrionis and kurstaki and the antibodies that they induce. This method is more sensitive and rapid than the dot-blot ELISA, and processing of many samples is easily accomplished.Key words: flow cytometry, soil, insecticidal toxins, Bacillus thuringiensis, clay, silt.

scholarly journals Immunofluorescence Flow Cytometry Technique for Enumeration of the Brown-Tide Alga, Aureococcus anophagefferens

2008 ◽  
Vol 74 (22) ◽  
pp. 6931-6940 ◽  
Author(s):  
Beth A. Stauffer ◽  
Rebecca A. Schaffner ◽  
Catherine Wazniak ◽  
David A. Caron
Keyword(s):  
Flow Cytometry ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Brown Tide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Aureococcus Anophagefferens ◽  
Elisa Method ◽  
Intact Cells ◽  
Microscopy Technique ◽  
Wide Range

ABSTRACT A new immunologically based flow cytometry (IFCM) technique was developed to enumerate Aureococcus anophagefferens, a small pelagophyte alga that is the cause of “brown tides” in bays and estuaries of the mid-Atlantic states along the U.S. coast. The method utilizes a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC-MAb) to label the surface of A. anophagefferens cells which are then detected and enumerated by using a flow cytometer. Optimal conditions for FITC-MAb staining, including solution composition, incubation times, and FITC-MAb concentrations, were determined. The FITC-MAb method was tested for cross-reactivity with nontarget, similarly sized, photoautotrophic protists, and the method was compared to an enzyme-linked immunosorbent assay (ELISA) using the same MAb. Comparisons of the IFCM technique to traditional microscopy enumeration of cultures and spiked environmental samples showed consistent agreement over several orders of magnitude (r 2 > 0.99). Comparisons of the IFCM and ELISA techniques for enumerating cells from a predation experiment showed a substantial overestimation (up to 10 times higher) of the ELISA in the presence of consumers of A. anophagefferens, presumably due to egested cell fragments that retained antigenicity, using the ELISA method, but were not characterized as whole algal cells by the IFCM method. Application of the IFCM method to environmental “brown-tide” samples taken from the coastal bays of Maryland demonstrated its efficacy in resolving A. anophagefferens abundance levels throughout the course of a bloom and over a large range of abundance values. IFCM counts of the brown-tide alga from natural samples were consistently lower than those obtained using the ELISA method and were equivalent to those of the polyclonal immunofluorescence microscopy technique, since both methods discriminate intact cells. Overall, the IFCM approach was an accurate and relatively simple technique for the rapid enumeration of A. anophagefferens in natural samples over a wide range of abundance values (103 to 106 cells ml−1).

scholarly journals Flow Cytometric Analysis of Microsporidia Belonging to the Genus Encephalitozoon

1999 ◽  
Vol 37 (2) ◽  
pp. 371-375 ◽  
Author(s):  
Delynn M. Moss ◽  
Gian P. Croppo ◽  
Sara Wallace ◽  
Govinda S. Visvesvara
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Polyclonal Antibodies ◽  
Flow Cytometric Analysis ◽  
Rabbit Antiserum ◽  
Fluorescein Isothiocyanate ◽  
Epidemiologic Studies ◽  
Cross Reactivity ◽  
Light Scatter ◽  
Flow Cytometric

Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-E. hellem serum was reactive to E. hellem spores but also had cross-reactivity to spores of E. cuniculi andE. intestinalis. On the other hand, fluorescence histograms showed that rabbit anti-E. cuniculi and rabbit anti-E. intestinalis sera were reactive with homologous spores only. Monoclonal antibody prepared against E. hellemreacted only with spores of E. hellem. Neither the polyclonal antibodies nor the monoclonal antibodies reacted withCryptosporidium parvum oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of Encephalitozoonwere identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of Encephalitozoon spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

Glycoconjugates on the surface of spores of the pathogenic fungus Phytophthora cinnamomi studied using fluorescence and electron microscopy and flow cytometry

1990 ◽  
Vol 36 (3) ◽  
pp. 183-192 ◽  
Author(s):  
A. R. Hardham ◽  
E. Suzaki
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Phytophthora Cinnamomi ◽  
Pathogenic Fungus ◽  
Fluorescein Isothiocyanate ◽  
Pathogenic Fungi ◽  
Surface Flow ◽  
Soybean Agglutinin ◽  
Binding Material

Glycoconjugates on the surface of zoospores and cysts of the pathogenic fungus Phytophthora cinnamomi have been studied using fluorescein isothiocyanate labelled lectins for fluorescence microscopy and flow cytometry, and ferritin- and gold-labelled lectins for ultrastructural analysis. Of the five lectins used, only concanavalin A (ConA) binds to the surface of the zoospores, including the flagella and water expulsion vacuole. This suggests that of accessible saccharides, glucosyl or mannosyl residues predominate on the outer surface of the zoospore plasma membrane. Early in encystment, a system of flat disc-like cisternae, which underlie the zoospore plasma membrane, vesiculate. These and other small peripheral vesicles quickly disappear. After the induction of encystment, ConA is no longer localised close to the plasma membrane but binds to material loosely associated with the cell surface. Quantitative measurements by flow cytometry indicate that the ConA-binding material is gradually lost from the cell surface. The cyst wall is weakly labelled, but the site of germ tube emergence stains intensely. During the first 2 min after the induction of encystment, material that binds soybean agglutinin, Helix pommatia agglutinin, and peanut agglutinin appears on the surface of the fungal cells. The distribution of this material, rich in galactosyl or N-acetyl-D-galactosaminosyl residues, is initially patchy, but by 5 min the material evenly coats most of the cell surface. Labelling of zoospores in which intracellular sites are accessible indicates that the soybean agglutinin binding material is stored in vesicles that lie beneath the plasma membrane. Quantitation of soybean agglutinin labelling shows that maximum binding occurs 2–3 min after the induction of encystment. Key words: cell surface, flow cytometry, lectins, pathogenic fungi, Phytophthora cinnamomi.

scholarly journals COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR

1996 ◽  
Vol 42 (12) ◽  
pp. 1915-1923 ◽  
Author(s):  
N DiDomenico ◽  
H Link ◽  
R Knobel ◽  
T Caratsch ◽  
W Weschler ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Internal Control ◽  
Detection System ◽  
Dna Amplification ◽  
Cross Reactivity ◽  
Diagnostic Pcr ◽  
Thermal Cycler ◽  
Paramagnetic Microparticles ◽  
Rna And Dna ◽  
Single Reaction

Abstract The COBAS AMPLICOR system automates amplification and detection of target nucleic acids, making diagnostic PCR routine for a variety of infectious diseases. The system contains a single thermal cycler with two independently regulated heating/cooling blocks, an incubator, a magnetic particle washer, a pipettor, and a photometer. Amplified products are captured on oligonucleotide-coated paramagnetic microparticles and detected with use of an avidin-horseradish peroxidase (HRP) conjugate. Concentrated solutions of amplicon or HRP were pipetted without detectable carryover. Amplified DNA was detected with an intraassay CV of < 4.5%; the combined intraassay CV for amplification and detection was < 15%. No cross-reactivity was observed when three different target nucleic acids were amplified in a single reaction and detected with three target-specific capture probes. The initial COBAS AMPLICOR menu includes qualitative tests for diagnosing infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and hepatitis C virus. All tests include an optional Internal Control to provide assurance that specimens are successfully amplified and detected.

scholarly journals Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19

2021 ◽  
Vol 22 (5) ◽  
pp. 2723
Author(s):  
Linhua Tian ◽  
Elzafir B. Elsheikh ◽  
Paul N. Patrone ◽  
Anthony J. Kearsley ◽  
Adolfas K. Gaigalas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Epidemiologic Studies ◽  
Cross Reactivity ◽  
Receptor Binding Domain ◽  
Proof Of Concept ◽  
Reference Standard ◽  
Improve Measurement ◽  
Antibody Titers

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

scholarly journals When brain damage “improves” perception: neglect patients can localize motion-shifted probes better than controls

2015 ◽  
Vol 114 (6) ◽  
pp. 3351-3358 ◽  
Author(s):  
Stefania de Vito ◽  
Marine Lunven ◽  
Clémence Bourlon ◽  
Christophe Duret ◽  
Patrick Cavanagh ◽  
...  
Keyword(s):  
Right Hemisphere ◽  
Visual Fields ◽  
Critical Factor ◽  
Significant Shift ◽  
Stimulus Motion ◽  
Attentional Processes ◽  
Position Shift ◽  
The Right ◽  
High Level ◽  
Better Than

When we look at bars flashed against a moving background, we see them displaced in the direction of the upcoming motion (flash-grab illusion). It is still debated whether these motion-induced position shifts are low-level, reflexive consequences of stimulus motion or high-level compensation engaged only when the stimulus is tracked with attention. To investigate whether attention is a causal factor for this striking illusory position shift, we evaluated the flash-grab illusion in six patients with damaged attentional networks in the right hemisphere and signs of left visual neglect and six age-matched controls. With stimuli in the top, right, and bottom visual fields, neglect patients experienced the same amount of illusion as controls. However, patients showed no significant shift when the test was presented in their left hemifield, despite having equally precise judgments. Thus, paradoxically, neglect patients perceived the position of the flash more veridically in their neglected hemifield. These results suggest that impaired attentional processes can reduce the interaction between a moving background and a superimposed stationary flash, and indicate that attention is a critical factor in generating the illusory motion-induced shifts of location.

Abstract T P238: Microglial CX3CR1 Required for M2 Polarization and Recovery After Intracerebral Hemorrhage

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Roslyn A Taylor ◽  
Matthew D Hammond ◽  
Youxi Ai ◽  
Lauren H Sansing
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Intracerebral Hemorrhage ◽  
Gait Analysis ◽  
Functional Outcome ◽  
Cylinder Test ◽  
M2 Microglia ◽  
Inflammatory Phenotype ◽  
Peripheral Leukocytes ◽  
The Right

Introduction: Intracerebral hemorrhage (ICH) results in the activation of microglia, the resident immune cells of the central nervous system. Microglia may polarize into an M1, pro-inflammatory phenotype, or an M2 phenotype associated with repair. CX3CR1 is a chemokine receptor on microglia and monocyte subsets. CX3CR1-null microglia have been shown to have dysregulated inflammation. We hypothesize that CX3CR1-null microglia have a prolonged M1 phenotype, contributing to worse functional outcome after ICH. Methods: ICH was modeled by injection of 20μl of blood into the right striatum. Neurological deficit was quantified using digital gait analysis, cylinder test, and beam walking. Mice were sacrificed 14 days after ICH; brains were harvested for flow cytometry and immunohistochemistry (IHC). C57BL/6 (WT) and CX3CR1 GFP/GFP (CX3CR1-null) mice were irradiated and reconstituted with bone marrow from WT mice carrying the congenic marker CD45.1 to generate bone marrow chimeras (CD45.1WT or CD45.1CX3CR1-null). M1 microglia were identified as expressing MHCII and M2 microglia with CD206. Results: The CD45.1CX3CR1-null mice show worse functional outcome 14 days after ICH by cylinder test (p=0.002), beam walking (p=<0.001) and gait analysis (p=0.02). By flow cytometry, few peripheral leukocytes remain in the brain at 14 days, indicating that F4/80 + and CD11b + cells visualized by IHC are likely microglia, not peripheral macrophages. By IHC, CD45.1 CX3CR1-null mice have significantly more amoeboid F4/80 + MHCII + cells per field (M1 microglia) than CD45.1WT mice (p=0.02). CD45.1 CX3CR1-null mice have significantly fewer CD11b + CD206 + cells per field (M2 microglia) compared to CD45.1WT mice (p=0.04). Conclusions: Our results suggest microglial CX3CR1 signaling is necessary for microglia to transition from M1 to M2 and contribute to recovery after ICH.

scholarly journals Transcriptional Analysis of the Toxin-Coding Plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis

2006 ◽  
Vol 72 (3) ◽  
pp. 1771-1776 ◽  
Author(s):  
Claudia Stein ◽  
Gareth W. Jones ◽  
Tanya Chalmers ◽  
Colin Berry
Keyword(s):  
Gene Regulation ◽  
Bacillus Thuringiensis ◽  
Transcriptional Regulators ◽  
Transcriptional Analysis ◽  
Host Bacterium ◽  
Large Plasmid ◽  
Coding Sequences ◽  
Physiological Processes ◽  
Insecticidal Toxins

ABSTRACT In Bacillus thuringiensis subsp. israelensis all of the insecticidal toxins are encoded on a single, large plasmid, pBtoxis. Sequencing of this plasmid revealed 125 potential coding sequences, many of which have predicted functions in gene regulation and physiological processes, such as germination. As a first step in understanding the possible role of pBtoxis in its host bacterium, a survey of the transcription of genes with predicted functions was carried out. Whereas many coding sequences, including those previously identified as probable pseudogenes, were not transcribed, mRNA was detected for 29 of the 40 sequences surveyed. Several of these sequences, including eight with similarities to the sequences of known transcriptional regulators, may influence wider gene regulation and thus may alter the phenotype of the host bacterium.

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