scholarly journals Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera

1998 ◽  
Vol 44 (3) ◽  
pp. 502-508 ◽  
Author(s):  
Gerhard Küllertz ◽  
Sabine Lüthe ◽  
Gunter Fischer
Keyword(s):  
Biological Fluids ◽  
Microtiter Plate ◽  
Serum Samples ◽  
Ppiase Activity ◽  
Microtiter Plate Assay ◽  
Isomerase Activity ◽  
Microtiter Plates ◽  
Activity Factor ◽  
Healthy Donors ◽  
Human Sera

Abstract An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay’s capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann–Whitney rank-sum test P <0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 °C was stable for at least 20 h.

Plasma Tyrosine Kinase Activity as A Potential Biomarker In BCR-ABL1-Targeted Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2738-2738
Author(s):  
Chen-Hsiung Yeh ◽  
Adam Abdool ◽  
Jean-marie Bruey
Keyword(s):  
Targeted Therapy ◽  
Tyrosine Kinase ◽  
Ex Vivo ◽  
Transcript Level ◽  
Microtiter Plate ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Serum Samples ◽  
Healthy Donors ◽  
Potential Biomarker

Abstract Abstract 2738 Introduction: In targeted therapy using tyrosine kinase (TK) inhibitors (TKIs), measurement of TK activity could help in diagnosis, identification of potential responders, and monitoring of treatment efficacy. Here we evaluated the feasibility of measuring circulating TK (cTK) activity in plasma in patients with BCR-ABL1-positive leukemia. Patients and Methods: Study subjects included 46 patients with newly diagnosed chronic myelogenous leukemia (CML), 24 with multidrug-resistant CML, 24 with BCR-ABL1-positive acute lymphocytic leukemia (ALL), as well as 38 healthy donors. Plasma cTK activity was measured using a chromogenic universal TK assay in which plasma samples and standards were added to wells of a microtiter plate that were coated with synthesized poly(Glu-Tyr) peptide substrates. Specific TK activity was measured by spectrophotometry, with or without addition of TKIs. Results: cTK activity was significantly higher in CML (median 801.93 U/mL, range 18.10–3932.30 U/mL) and BCR-ABL1-positive ALL patients (median 659.55 U/mL, range 0–1626.90 U/mL) than in healthy donors (median 82.85 U/mL, range 0.63–852.80 U/mL) (P<0.001). Plasma cTK activity was closely correlated with cellular BCR-ABL1 kinase activation, as indicated by phosphorylation of the downstream signaling proteins CRKL (P<0.001) and STAT-5 (P=0.003). However, cTK activity was independent of BCR-ABL1 transcript level and BCR-ABL1 mutation type. Plasma cTK activity was inhibited by imatinib and dasatinib in ex vivo studies; this inhibition was severely diminished in patients harboring T315I mutations. Conclusions: cTK activity is readily detectable in plasma or serum samples without pre-cleaning or enrichment. Ex vivo testing measuring the effect of TKIs on plasma cTK activity thus hold promise as drug sensitivity tests for predicting and monitoring response to specific TKIs. Disclosures: Yeh: Quest Diagnostics Inc.: Employment. Abdool:Quest Diagnostics Inc.: Employment. Bruey:Quest Diagnostics Inc.: Employment.

A NOVEL MICROTITER PLATE ASSAY FOR FUNCTIONAL PLASMINOGEN-ACTIVA-TOR INHIBITOR (PAI) LEVELS IN WHOLE PLASMA

1987 ◽  
Author(s):  
R C Carroll ◽  
R M HAnsard ◽  
M H GoldMan
Keyword(s):  
Age Groups ◽  
Microtiter Plate ◽  
Functional Assay ◽  
Age Group ◽  
Plasma Samples ◽  
Major Drawback ◽  
Microtiter Plate Assay ◽  
Microtiter Plates ◽  
Linear Slope ◽  
Plasmin Inhibitor

Currently, the most widely used functional assay for PAI levels in plasma is that developed by Chmielewska et al (Thromb. Res. 31:427-436, 1983). A major drawback of this assay is the considerable manipulation of plasma samples required by the necessity of acidification and reneutralization in order to determine remaining activator levels based on plasminogen to plasmin conversion. We have devised an assay based on microtiter plates coated with 20 U/ml urokinase. In this assay, aliquots of citrate- or EDTA-anticoagulated plasma are incubated in the coated wells for 30 min at room temperature to allow PAI in the plasma to complex with the solid state urokinase. The plasma is then removed, the wells extensively washed with Tris-saline buffer plus 1 mg/ml bovine serum albumin, and the remaining urokinase activity determined by adding plasminogen and S-2251 chroma-genic substrate for plasmin. Inhibitor levels can be conveniently expressed as percent inhibition relative to the urokinase activity in wells not exposed to plasma. Absolute units of inhibitor have also been derived by titration of plasma samples with known units of urokinase (calibrated by S-2444 chromagenic substrate for plasminogen activators). Between the range of 90% to 10% inhibition, a linear slope of 0.69±0.6 units ml-1/10% inhibition is obtained.In agreement with previous reports, we find a wide variation in control subjects and some tendency towards higher PAI levels in older age groups. Age group 20-39, n=29, 36.6 ± 14.9%. Age group 40 and over, n=16, 48.9 ±12.9%.Reproducible values (SEM ± 4.6%,n=5)are obtained for control* individuals sampled and assayed at various times over a period of several months. We have also assayed plasma samples from patients undergoing a variety of surgical procedures, and have followed rapid post-surgical increases in PAI levels with a gradual return to pre-operative levels. Evidence is presented that the post-surgical increase is predominantly from the platelet releasable pool.

Hydrodynamic Evaluation of Microtiter Plate Assay Using Computational Fluid Dynamics for Biofilm Formation

2019 ◽  
Author(s):  
R. Zahra ◽  
A. A. Khan ◽  
M. Sajid
Keyword(s):  
Fluid Dynamics ◽  
Computational Fluid Dynamics ◽  
Biofilm Formation ◽  
Microtiter Plate ◽  
Bacterial Cells ◽  
Microtiter Plate Assay ◽  
Plate Assay ◽  
Microtiter Plates ◽  
Biofilm Production ◽  
Static Conditions

Abstract Biofilms are complex surface associated communities where bacterial cells are enclosed by self-produced extra cellular polymeric substances (EPS), mainly consisting of exopolysaccharides, proteins and extracellular DNA. Treatment of biofilm associated persistent infections is an emerging issue for clinicians as bacterial cells adhere with human epithelial cells or indwelling medical devices such as implants and catheters, used in urinary tract and respiratory infections. Several methods are in practice to assess the biofilm formation of bacterial strains. Most of these are phenotypic methods which include Congo red assay (CRA), Air liquid interface (ALI), tissue culture plate method and Microtiter plate assay (MTPA). MTPA is considered as a standard screening method for comparing adherence pattern and is the most widely used quantitative method for detection of biofilm formation. Generally, the assay is performed under standard static conditions and little is known about the hydrodynamics in the microtiter plates. A few studies have applied computational fluid dynamics (CFD) simulations to describe flow pattern in microtiter plates during biofilm production and optimized the suitable conditions to detect the biofilm formation which have proven to be efficient. In this work the dependencies of biofilm formation on the hydrodynamics in microtiter plate assays were evaluated using OpenFOAM® an open-source toolbox for numerical simulation. It was found that higher flow rates increase the nutrient availability, promote cell growth, and attachment pattern with increased production of exopolymer, while the increase in flow velocity increases the shear rate causing erosion and disassembly of biofilm production because of detachment from the surface.

scholarly journals Detection of Human Herpesvirus 6 DNA in Serum by a Microplate PCR-Hybridization Assay

1998 ◽  
Vol 36 (1) ◽  
pp. 68-72 ◽  
Author(s):  
Carla Osiowy ◽  
Isabelle Prud’homme ◽  
Michael Monette ◽  
Shimian Zou
Keyword(s):  
Human Herpesvirus ◽  
Microtiter Plate ◽  
High Rate ◽  
Human Herpesvirus 6 ◽  
Serum Samples ◽  
Microtiter Plate Assay ◽  
Serological Examination ◽  
Serological Status ◽  
Serum Dna ◽  
Herpesvirus 6

PCR was performed on DNA extracts derived from clinical serum samples submitted for human herpesvirus 6 (HHV-6) serological examination. To detect amplified HHV-6 products, a hybridization-based microtiter plate assay (PCR ELISA; Boehringer Mannheim) was used. The assay system was found to be rapid, specific, and sensitive. Approximately three copies of a plasmid-based HHV-6 sequence could be detected, and no cross amplification was observed with HHV-7 genomic DNA. There was no correlation found between HHV-6 DNA detection and serological status in clinical serum samples from individuals more than 2 years old. On the other hand, in serum samples from infants less than 2 years old, a high rate of detection of HHV-6 DNA was observed in those who lacked immunoglobulin G and M antibodies to HHV-6 (55%). In this regard, PCR of serum DNA extracts may be used as a sensitive indicator of active HHV-6 infection in infants prior to their seroconversion.

A monoclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein.

1987 ◽  
Vol 33 (12) ◽  
pp. 2275-2277 ◽  
Author(s):  
N H Fraeyman ◽  
E J Van de Velde ◽  
F H De Smet
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Biological Fluids ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rabbit Antibody ◽  
Acid Glycoprotein ◽  
Microtiter Plates ◽  
Sandwich Type ◽  
Human Sera

Abstract A "sandwich"-type enzyme-linked immunosorbent assay for determining concentrations of human alpha 1-acid glycoprotein (AGP) is described. Microtiter plates coated with a polyclonal rabbit antibody to human AGP were subsequently incubated with the antigen, with a specific murine monoclonal antibody, and with goat anti-mouse immunoglobulins conjugated to alkaline phosphatase. To evaluate the method for assay of AGP in human sera, we compared it with single radial immunodiffusion and "rocket" electroimmunoassay. The respective correlations were r = 0.988 (n = 45) and r = 0.973 (n = 47). Repeated assays of a human serum sample with an average AGP concentration of 859 mg/L yielded within-day and between-day CVs of 1.4% (n = 5) and 6.3% (n = 10), respectively. Because of its low detection limit (4.4 micrograms/L), this assay is also suitable for determination of AGP concentrations in other biological fluids, such as dialysates of patients being treated by continuous ambulatory peritoneal dialysis.

scholarly journals Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1

2002 ◽  
Vol 68 (6) ◽  
pp. 2683-2689 ◽  
Author(s):  
Anders R. Johnsen ◽  
Karen Bendixen ◽  
Ulrich Karlson
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
Aromatic Hydrocarbons ◽  
Carbon Sources ◽  
Microtiter Plate ◽  
Water Soluble ◽  
Most Probable Number ◽  
Microtiter Plate Assay ◽  
Probable Number ◽  
Microtiter Plates ◽  
Polycyclic Aromatic

ABSTRACT We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.

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

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.

scholarly journals Surface Adsorption of the Cancer Biomarker Lysophosphatidic Acid in Serum Studied by Acoustic Wave Biosensor

Materials ◽  
2021 ◽  
Vol 14 (15) ◽  
pp. 4158
Author(s):  
Brian De La Franier ◽  
Michael Thompson
Keyword(s):  
Acoustic Wave ◽  
Lysophosphatidic Acid ◽  
Electrode Surface ◽  
Biological Fluids ◽  
Thiol Group ◽  
Cancer Biomarker ◽  
Specific Adsorption ◽  
Shear Mode ◽  
Serum Samples ◽  
Fouling Reduction

The thickness shear mode acoustic wave device is of interest for the sensing of biomarkers for diseases in various biological fluids, but suffers from the issue of non-specific adsorption of compounds other than those of interest to the electrode surface, thus affecting the device’s output. The aim of this present study was to determine the level of non-specific adsorption on gold electrodes from serum samples with added ovarian cancer biomarker lysophosphatidic acid in the presence of a surface anti-fouling layer. The latter was an oligoethylene molecule with thiol group for attachment to the electrode surface. It was found that the anti-fouling layer had a minimal effect on the level of both adsorption of components from serum and the marker. This result stands in sharp contrast to the analogous monolayer employed for anti-fouling reduction on silica.

scholarly journals Antibodies to Highly Pathogenic A/H5Nx (Clade 2.3.4.4) Influenza Viruses in the Sera of Vietnamese Residents

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 394
Author(s):  
Tatyana Ilyicheva ◽  
Vasily Marchenko ◽  
Olga Pyankova ◽  
Anastasia Moiseeva ◽  
Tran Thi Nhai ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Wide Spectrum ◽  
Influenza Viruses ◽  
Serum Samples ◽  
Socialist Republic ◽  
Highly Pathogenic ◽  
Virus Variant ◽  
Human Sera ◽  
Hemagglutinin Inhibition

To cause a pandemic, an influenza virus has to overcome two main barriers. First, the virus has to be antigenically new to humans. Second, the virus has to be directly transmitted from humans to humans. Thus, if the avian influenza virus is able to pass the second barrier, it could cause a pandemic, since there is no immunity to avian influenza in the human population. To determine whether the adaptation process is ongoing, analyses of human sera could be conducted in populations inhabiting regions where pandemic virus variant emergence is highly possible. This study aimed to analyze the sera of Vietnamese residents using hemagglutinin inhibition reaction (HI) and microneutralization (MN) with A/H5Nx (clade 2.3.4.4) influenza viruses isolated in Vietnam and the Russian Federation in 2017–2018. In this study, we used sera from 295 residents of the Socialist Republic of Vietnam collected from three groups: 52 samples were collected from households in Nam Dinh province, where poultry deaths have been reported (2017); 96 (2017) and 147 (2018) samples were collected from patients with somatic but not infectious diseases in Hanoi. In all, 65 serum samples were positive for HI, at least to one H5 virus used in the study. In MN, 47 serum samples neutralizing one or two viruses at dilutions of 1/40 or higher were identified. We postulate that the rapidly evolving A/H5Nx (clade 2.3.4.4) influenza virus is possibly gradually adapting to the human host, insofar as healthy individuals have antibodies to a wide spectrum of variants of that subtype.

Development of an HPLC method for measuring orally administered 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in biological fluids

1990 ◽  
Vol 36 (1) ◽  
pp. 5-8 ◽  
Author(s):  
J G Goddard ◽  
G J Kontoghiorghes
Keyword(s):  
High Performance ◽  
Biological Fluids ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Hplc Method ◽  
Iron Chelators ◽  
Serum Samples ◽  
Thalassemic Patient ◽  
Serum And Urine

Abstract "High-performance" liquid-chromatographic (HPLC) methods have been developed for identifying 1-substituted 2-alkyl-3-hydroxypyrid-4-one iron chelators in serum and urine. Ion pairing with heptane- or octanesulfonic acid in pH 2.0-2.2 phosphate buffer and reversed-phase chromatography were required to separate these compounds from endogenous compounds in both biological fluids. In both the 2-methyl and 2-ethyl series of 1-substituted compounds (H, methyl, ethyl, or propyl) the elution times increased in accordance with the n-octanol/water partition coefficients (propyl greater than ethyl greater than H greater than methyl). Urine samples were filtered (0.4 microns pore size) and injected either undiluted or after dilution with elution buffer. After the addition of internal standard, the plasma or serum samples were deproteinized by treatment with HCIO4, 0.5 mol/L, centrifuged, and the supernates were injected directly onto the HPLC. Using these procedures, we could identify 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in the serum and urine of a thalassemic patient who had received a 3-g dose of the drug and in the urine of other patients who had received the same dose. One or more possible metabolites were also observed in the chromatograms of both urine and serum. The 24-h urinary output of L1 (0.22-2.37 g) and iron (10.6-71.5 mg) varied but there was no correlation between the two with respect to quantity or concentration. Instead, urinary iron output was higher in patients with a greater number of transfused units of erythrocytes. This is the first study in humans to show that L1 is absorbed from the gut, enters the circulation, and is excreted in the urine.

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