A sensitive pH indicator-based spectrophotometric assay for PHB depolymerase activity on microtiter plates

Maria Angeles Camacho-Ruiz; Marcelo Müller-Santos; Xitlalli D. Hernández-Mancillas; Vicente Paul Armenta-Perez; Edgar Zamora-Gonzalez; Jorge A. Rodríguez

doi:10.1039/d0ay00840k

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159436 ◽

1990 ◽

Vol 48 (3) ◽

pp. 378-379

Author(s):

Jane E. Ramberg ◽

Shigeto Tohma ◽

Peter E. Lipsky

Keyword(s):

B Cells ◽

Adhesion Molecule ◽

Colloidal Gold ◽

Intercellular Adhesion ◽

Intercellular Adhesion Molecule ◽

Staining Procedure ◽

Double Staining ◽

T And B Cells ◽

Microtiter Plates ◽

Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

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Prevalence and Induction of Circulating Antibodies against Recombinant Staphylokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642396 ◽

1994 ◽

Vol 71 (01) ◽

pp. 129-133 ◽

Cited By ~ 33

Author(s):

P J Declerck ◽

S Vanderschueren ◽

J Billiet ◽

H Moreau ◽

D Collen

Keyword(s):

Intravenous Administration ◽

Human Subjects ◽

Microtiter Plates ◽

Staphylococcus Aureus Bacteremia ◽

Alternative Agent ◽

Antibody Titers ◽

Potential Alternative ◽

Circulating Antibodies ◽

Low Levels ◽

Antibody Levels

SummaryStreptokinase (SK) is a routinely used thrombolytic agent but it is immunogenic and allergenic; staphylokinase (STA) is a potential alternative agent which is under early clinical evaluation. The comparative prevalence of antibodies against recombinant STA (STAR) and against SK was studied in healthy subjects and their induction with intravenous administration in small groups of patients.Enzyme-linked immunosorbent assays, using microtiter plates coated with STAR or SK and calibration with affinospecific human antibodies, revealed 2.1 to 65 μg/ml (median 11 μg/ml) anti-STAR antibodies and 0.9 to 370 μg/ml (median 18 μg/ml) anti-SK antibodies (p <0.001 vs anti-STAR antibodies) in plasma from 100 blood donors, with corresponding values of 0.6 to 100 μg/ml (median 7.1 μg/ml) and 0.4 to 120 μg/ml (median 7.3 μg/ml), respectively, in 104 patients with angina pectoris. Three out of 17 patients with Staphylococcus aureus bacteremia had significantly increased anti-STAR antibody levels (150, 75 and 75 μg/ml), and STAR neutralizing activities (2.2, 3.6 and 4.1 μg STAR neutralized per ml plasma, respectively). In 6 patients with acute myocardial infarction, given 10 mg STAR intravenously over 30 min, median anti-STAR antibody levels were 3.5 μg/ml at baseline, 2.9 μg/ml at 6 to 8 days and 1.2 μg/ml at 2 to 9 weeks, with median corresponding titers of STAR neutralizing activity at 2 to 9 weeks of 42 μg/ml plasma. Conversely, in 5 patients treated with 1,500,000 units SK over 60 min, median anti-SK antibodies increased from 2.9 μg/ml at baseline to 360 μg/ml at 5 to 10 days, with corresponding median SK neutralizing activities of 13 μg/ml. Antibodies against STAR did not cross-react with SK and vice versa.Plasma from human subjects contains low levels of circulating antibodies against recombinant staphylokinase, and intravenous administration of this compound boosts antibody titers. These antibodies do however not cross-react with streptokinase, whereby the use of these two immunogenic thrombolytic agents would not be mutually exclusive.

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Visualizing Preferential Flow Paths using Ammonium Carbonate and a pH Indicator

Soil Science Society of America Journal ◽

10.2136/sssaj2002.0347 ◽

2002 ◽

Vol 66 (2) ◽

pp. 347 ◽

Cited By ~ 10

Author(s):

Zhi Wang ◽

Jianhang Lu ◽

Laosheng Wu ◽

Thomas Harter ◽

William A. Jury

Keyword(s):

Preferential Flow ◽

Ammonium Carbonate ◽

Ph Indicator ◽

Flow Paths ◽

Preferential Flow Paths

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Suitability of the Cyclic Voltammetry Measurements and DPPH• Spectrophotometric Assay to Determine the Antioxidant Capacity of Food-Grade Oenological Tannins

Molecules ◽

10.3390/molecules24162925 ◽

2019 ◽

Vol 24 (16) ◽

pp. 2925 ◽

Cited By ~ 7

Author(s):

Arianna Ricci ◽

Giuseppina Paola Parpinello ◽

Nemanja Teslić ◽

Paul Andrew Kilmartin ◽

Andrea Versari

Keyword(s):

Cyclic Voltammetry ◽

Colorimetric Assay ◽

Radical Scavenging ◽

Calibration Graph ◽

Spectrophotometric Assay ◽

Experimental Conditions ◽

Fast Analysis ◽

Cv Measurements ◽

And Control ◽

Analytical Approaches

Twenty commercially available oenological tannins (including hydrolysable and condensed) were assessed for their antiradical/reducing activity, comparing two analytical approaches: The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging spectrophotometric assay and the cyclic voltammetry (CV) electrochemical method. Electrochemical measurements were performed over a −200 mV–500 mV scan range, and integrated anodic currents to 500 mV were used to build a calibration graph with (+)-catechin as a reference standard (linear range: From 0.0078 to 1 mM, R2 = 0.9887). The CV results were compared with the DPPH• assay (expressed as % of radical scavenged in time), showing high correlation due to the similarity of the chemical mechanisms underlying both methods involving polyphenolic compounds as reductants. Improved correlation was observed by increasing the incubation time with DPPH• to 24 h (R2 = 0.925), demonstrating that the spectrophotometric method requires a long-term incubation to complete the scavenging reaction when high-molecular weight tannins are involved; this constraint has been overcome by using instant CV measurements. We concluded that the CV represents a valid alternative to the DPPH• colorimetric assay, taking advantage of fast analysis and control on the experimental conditions and, because of these properties, it can assist the quality control along the supply chain.

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Fully Automated Parallel Oligonucleotide Synthesizer

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20011299 ◽

2001 ◽

Vol 66 (8) ◽

pp. 1299-1314 ◽

Cited By ~ 14

Author(s):

Michal Lebl ◽

Christine Burger ◽

Brett Ellman ◽

David Heiner ◽

Georges Ibrahim ◽

...

Keyword(s):

Solid Phase ◽

Building Blocks ◽

Solid Support ◽

Continuous Operation ◽

Gear Pump ◽

Oligonucleotide Synthesis ◽

Center Of Rotation ◽

Microtiter Plates ◽

The Individual ◽

Design And Construction

Design and construction of automated synthesizers using the tilted plate centrifugation technology is described. Wash solutions and reagents common to all synthesized species are delivered automatically through a 96-channel distributor connected to a gear pump through two four-port selector valves. Building blocks and other specific reagents are delivered automatically through banks of solenoid valves, positioned over the individual wells of the microtiterplate. These instruments have the following capabilities: Parallel solid-phase oligonucleotide synthesis in the wells of polypropylene microtiter plates, which are slightly tilted down towards the center of rotation, thus generating a pocket in each well, in which the solid support is collected during centrifugation, while the liquid is expelled from the wells. Eight microtiterplates are processed simultaneously, providing thus a synthesizer with a capacity of 768 parallel syntheses. The instruments are capable of unattended continuous operation, providing thus a capacity of over two millions 20-mer oligonucleotides in a year.

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Regression Analysis of Orthogonal, Cylindrical and Multivariable Color Parameters for Colorimetric Surface pH Measurement of Materials

Molecules ◽

10.3390/molecules26123682 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3682

Author(s):

Katarína Vizárová ◽

Izabela Vajová ◽

Naďa Krivoňáková ◽

Radko Tiňo ◽

Zdenko Takáč ◽

...

Keyword(s):

Ph Value ◽

Critical Factor ◽

Ph Measurement ◽

Ph Indicator ◽

Water Quality Control ◽

Quality Management Systems ◽

Surface Ph ◽

Regression Parameters ◽

Ph Detection

The surface pH is a critical factor in the quality and longevity of materials and products. Traditional fast colorimetric pH detection-based tests such as water quality control or pregnancy tests, when results are determined by the naked eye, cannot provide quantitative values. Using standard pH papers, paper-printed comparison charts, or colorimetric microfluidic paper-based analytical devices is not suitable for such technological applications and quality management systems (QMSs) where the particular tested material should contain a suitable indicator in situ, in its structure, either before or after the process, the technology or the apparatus that are being tested. This paper describes a method based on the combination of impregnation of a tested material with a pH indicator in situ, its exposure to a process of technology whose impact on pH value is to be tested, colorimetric pH measurement, and approximation of pH value using derived pH characteristic parameters (pH-CPs) based on CIE orthogonal and cylindrical color variables. The hypotheses were experimentally verified using the methyl red pH indicator, impregnating the acid lignin-containing paper, and preparing a calibration sample set with pH in the range 4 to 12 using controlled alkalization. Based on the performed measurements and statistical evaluation, it can be concluded that the best pH-CPs with the highest regression parameters for pH are √∆E, ln (a),√∆H (ab), a/L, h/b and ln (b/a). The experimental results show that the presented method allows a good estimation of pH detection of the material surfaces.

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High-Throughput Peptide Derivatization toward Supramolecular Diversification in Microtiter Plates

ACS Nano ◽

10.1021/acsnano.0c05423 ◽

2021 ◽

Author(s):

Yiyang Lin ◽

Matthew Penna ◽

Christopher D. Spicer ◽

Stuart G. Higgins ◽

Amy Gelmi ◽

...

Keyword(s):

High Throughput ◽

Microtiter Plates ◽

Peptide Derivatization

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Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

Microbial Cell Factories ◽

10.1186/s12934-021-01541-2 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Tobias Habicher ◽

Tobias Klein ◽

Jacqueline Becker ◽

Andreas Daub ◽

Jochen Büchs

Keyword(s):

Controlled Release ◽

Bacillus Licheniformis ◽

Protease Activity ◽

Microtiter Plate ◽

Fed Batch ◽

Batch Mode ◽

Screening Process ◽

Microtiter Plates ◽

Batch Fermenter ◽

Production Conditions

Abstract Background Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. Results To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. Conclusion The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

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