DEVELOPMENT OF A TEST-SYSTEMS FOR THE IDENTIFICATION OF THE SOIL BORNED TOBACCO RATTLE VIRUS BASED ON THE USE OF ELISA

2019 ◽  
Vol 1 (1) ◽  
pp. 22-37
Author(s):  
Yury Varicev ◽  
Aleksander Uskov ◽  
Galina Variceva ◽  
Pavel Galushka ◽  
Yury Prihidko
Keyword(s):  
Sucrose Concentration ◽  
Tobacco Rattle Virus ◽  
Elisa Test ◽  
Virus Preparation ◽  
Test Systems ◽  
Ionic Detergent ◽  
Triton X ◽  
Polyethylene Glycol 6000 ◽  
Leaf Juice

On the base of the isolate TRV PV-0361 of the tobacco rattle virus from the commercial collection of the DSMZ company (Germany) have been developed methods for maintaining the virus in vitro culture on N. clevelandii plants, propagation the virus in the spring-summer period in greenhouse conditions, “soft” isolation of a purified virus preparation by clarifying leaf juice with low-speed centrifugation and treatment with non-ionic detergent Triton-X-100 followed by precipitation of the virus with polyethylene glycol-6000 and three-fold ultracentrifugation using sucrose cushion, sucrose concentration gradient and differential centrifugation. The purified virus preparation was used for producing rabbit antiserums according to the scheme we worked out. The obtained antiserum had the following titers - specific 1: 5 · 105, non-specific 1: 8 · 103. Based on antibodies isolated from this antiserum, coating antibodies and peroxidase conjugates were obtained, which made it possible to create ELISA test systems for determining TRV with sensitivity of about 12-16 ng/ml. The resulting test systems can be used in practical work on quality control and certification of seed potatoes.

scholarly journals Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma

1975 ◽  
Vol 152 (1) ◽  
pp. 143-151 ◽  
Author(s):  
C N Murty ◽  
E Verney ◽  
H Sidransky
Keyword(s):  
Protein Synthesis ◽  
Initiation Factors ◽  
Ionic Detergent ◽  
Membrane Bound ◽  
Triton X ◽  
Stimulation Of

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.

scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251 ◽  
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

scholarly journals The versatility of external quality assessment for the surveillance of laboratory and in vitro diagnostic performance: SARS-CoV-2 viral genome detection in Austria

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Christoph Buchta ◽  
Jeremy V. Camp ◽  
Jovana Jovanovic ◽  
Peter Chiba ◽  
Elisabeth Puchhammer-Stöckl ◽  
...  
Keyword(s):  
Quality Assessment ◽  
External Quality Assessment ◽  
False Negative ◽  
Positive Sample ◽  
Performance Criteria ◽  
External Quality ◽  
Assay Sensitivity ◽  
Dilution Series ◽  
Test Systems

Abstract Objectives External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. Methods Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. Results A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (C t ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of C t values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. Conclusions Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.

scholarly journals In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1784-1792 ◽  
Author(s):  
Gianluca Civenni ◽  
Samuel T. Test ◽  
Urs Brodbeck ◽  
Peter Bütikofer
Keyword(s):  
Membrane Vesicle ◽  
Human Erythrocytes ◽  
Membrane Extraction ◽  
Membrane Microdomains ◽  
Membrane Insertion ◽  
Vesicle Formation ◽  
Calcium Loading ◽  
Lipid Moiety ◽  
Triton X

Abstract In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

scholarly journals Cell Culture Based in vitro Test Systems for Anticancer Drug Screening

2020 ◽  
Vol 8 ◽  
Author(s):  
Kristina V. Kitaeva ◽  
Catrin S. Rutland ◽  
Albert A. Rizvanov ◽  
Valeriya V. Solovyeva
Keyword(s):  
Cell Culture ◽  
Anticancer Drug ◽  
Drug Screening ◽  
In Vitro Test ◽  
Test Systems ◽  
Vitro Test

scholarly journals Effects of detergent on the sulphation of chondroitin by cell-free preparations from chick-embryo epiphyseal cartilage

1992 ◽  
Vol 285 (2) ◽  
pp. 577-583 ◽  
Author(s):  
G Sugumaran ◽  
J E Silbert
Keyword(s):  
Chick Embryo ◽  
Supernatant Fraction ◽  
Random Pattern ◽  
Type I ◽  
Type Ii ◽  
Enzyme Preparations ◽  
Enzyme Substrate ◽  
Ionic Detergent ◽  
The Individual ◽  
Triton X

The effects of the non-ionic detergent Triton X-100 on 6-sulphation of two species of endogenous nascent proteochondroitin by a chick-embryo cartilage microsomal system was examined. Sulphation of the larger (Type I) species with adenosine 3′-phosphate 5′-phosphosulphate was slightly diminished when Triton X-100 was present, whereas sulphation of the smaller (Type II) species was slightly enhanced. An ordered rather than random pattern of sulphation was obtained for the smaller proteoglycan, but with a considerably lower degree of sulphation than that of the larger proteochondroitin. These differences were consistent with other differences between these two species as described previously. Sulphation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system with Triton X-100 present produced ordered rather than random sulphation patterns. When a 100,000 g supernatant fraction was utilized for sulphation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and ordered sulphation was still obtained. When hexasaccharide was used, sulphation of multiple N-acetylgalactosamine residues of the individual hexasaccharides resulted. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulphation of multiple N-acetylgalactosamine residues on the individual hexasaccharide molecules was even greater, so that tri-sulphated products were found. This suggests that ordered rather than random sulphation of chondroitin with these enzyme preparations is due to enzyme-substrate interaction rather than to membrane organization.

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

1991 ◽  
Vol 280 (3) ◽  
pp. 745-751 ◽  
Author(s):  
N M Hooper ◽  
A Bashir
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Aqueous Phase ◽  
Hydrophobic Membrane ◽  
Rich Phase ◽  
Glycosyl Phosphatidylinositol ◽  
Ionic Detergent ◽  
Membrane Spanning ◽  
Triton X ◽  
Insoluble Pellet

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

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