scholarly journals Qualitative Indicators of Experimental Brucellosis Antigen Preparations Designed for Cellular Tests in vitro

2020 ◽  
pp. 83-88
Author(s):  
S. A. Kurcheva ◽  
D. A. Kovalev ◽  
D. G. Ponomarenko ◽  
Yu. V. Siritsa ◽  
M. V. Kostyuchenko ◽  
...  
Keyword(s):  
Specific Activity ◽  
Laboratory Diagnosis ◽  
Polysaccharide Composition ◽  
Methodological Approaches ◽  
Cellular Tests ◽  
Refractometric Detection ◽  
Experimental Preparation ◽  
Selection Of

In order to develop the most diagnostically informative methods for carrying out antigen-stimulated cellular tests in vitro a careful selection of stimulating agent (antigen) is required, possessing an adequate activating potential and providing specificity of the reaction.Objective of the study was to identify the qualitative indicators of experimental batches of brucellosis antigen preparations designed for cellular tests in vitro.Materials and methods. Initially we produced antigen complexes of brucellosis microbe on the basis of the vaccine strains of three epidemically significant Brucella species (B. abortus, B. melitensis, B. suis). Quantitative determination of WsAg and PPBC proteins of experimental preparation series was performed applying capillary electrophoresis. Qualitative composition was assessed through ion exchange liquid chromatography with refractometric detection.Results and discussion. We have specified physical-chemical features, investigated chromatographic profiles and composition of protein fractions, as well as tried the produced experimental batches of brucellosis antigen preparations. After analyzing the defined protein and polysaccharide composition of the obtained WsAg samples, one can conclude that WsAg preparation cannot be used for cellular tests as the probability of non-specific lymphocyte reaction manifestation in vitro was experimentally proven. By contrast, complex brucellosis antigen preparation PPBC has an expressed specific activity and specificity under in vitro conditions and the prospects to be used when developing methodological approaches for laboratory diagnosis of brucellosis and assessment of de facto immunity rate in risk contingents after vaccination. The obtained parameters will allow for proper quality provision when manufacturing the developed experimental PPBC preparation designed for cellular tests in vitro, taking into account modern validation and standardization regulations. 

scholarly journals Quantification and characterization of the 5’exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay

2020 ◽  
pp. jbc.RA120.015867
Author(s):  
Cedric Cappel ◽  
Adriana Carolina Gonzalez ◽  
Markus Damme
Keyword(s):  
Lupus Erythematosus ◽  
Specific Activity ◽  
Nuclease Activity ◽  
Hydrolytic Activity ◽  
Proteolytic Processing ◽  
Exonuclease Activity ◽  
Fluorescence Quencher

Phospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer`s disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5’ exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher coupled oligonucleotides for the quantitative determination of acidic 5’ exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions, and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal acid 5’ exonuclease in HeLa cells, where it showed a markedly higher specific activity compared to PLD4. We further used our newly developed assay to determine the substrate specificity and inhibitory profile of PLD3, and found that proteolytic processing of PLD3 is dispensable for its hydrolytic activity. We followed the expression, proteolytic processing, and intracellular distribution of genetic PLD3 variants previously associated with Alzheimer’s disease and investigated each variant's effect on the 5’ nuclease activity of PLD3, finding that some variants lead to reduced activity, but others not. The development of a PLD3/4-specific biochemical assay will be instrumental in understanding better both nucleases and their incompletely unknown roles in vitro and in vivo.

scholarly journals Validation by isoelectric focusing of the anion-exchange isotransferrin fractionation step involved in determination of carbohydrate-deficient transferrin by the CDTect assay

1998 ◽  
Vol 44 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Torsten Arndt ◽  
Rolf Hackler ◽  
Tilman O Kleine ◽  
Axel M Gressner
Keyword(s):  
Isoelectric Focusing ◽  
Laboratory Diagnosis ◽  
Good Precision ◽  
Chronic Alcohol Abuse ◽  
Negative Results ◽  
Iron Saturation ◽  
Low Concentrations ◽  
Carbohydrate Deficient Transferrin

Abstract Serum concentration of carbohydrate-deficient transferrin (CDT) is used for laboratory diagnosis of chronic alcohol abuse. Using isoelectric focusing for validation of the initial isotransferrin fractionation step involved in the determination of CDT by the CDTect assay, we found a complete in vitro iron saturation of transferrin and sufficient stability of the transferrin iron load during column passage; effective separation of non-CDT-isotransferrins and CDT-isotransferrins at the microcolumns; partial coelution of trisialo-Fe2-transferrin, which did not significantly affect CDT measurement; partial retention of CDT-isotransferrins, especially disialo-Fe2-transferrin, which may cause falsely negative results for CDT at the upper reference limits; good precision of the isotransferrin fractionation step; and no significant effects of low concentrations of serum protein and transferrin. We strongly urge standardization of CDT analysis and suggest isoelectric focusing for validation of CDT analysis methods and verification of odd results.

scholarly journals METHODS FOR ASSESSMENT OF THE SPECIFIC ACTIVITY OF DRUGS FOR PREVENTION OF HEPATITIS B

2017 ◽  
Vol 62 (5) ◽  
pp. 233-240
Author(s):  
E. L. Postnova ◽  
N. V. Shalunova ◽  
K. A. Sarkisyan ◽  
A. A. Movsesyants
Keyword(s):  
Hepatitis B ◽  
Enzyme Immunoassay ◽  
Specific Activity ◽  
Elisa Method ◽  
Normative Documents ◽  
Immunologic Activity

The immunologic activity (specific activity) is one of the main indicators of quality of vaccines for prophylaxis of hepatitis B, along with their safety. Retrospective analysis of the use of laboratory methods for assessment of specific (immunogenic) activity of modern vaccines against hepatitis B using indicators was carried out: in vitro method based on evaluation of HBsAg content and in vivo method based on evaluation of immunogenic activity in mice. Both methods are standardized and described in normative documents on the vaccines against hepatitis B of domestic production registered in the Russian Federation. Indicators of specific (immunogenic) activity of vaccines against hepatitis B were used to investigate more than 170 vaccine series using the ELISA method in the period from 2013 to 2015. The obtained control results confirmed the expediency and efficiency of enzyme immunoassay for determination of HBsAg content, as well as permissibility of use of ready sets of the Murex HBsAg Version 3 test systems for testing vaccines against hepatitis B by the ELISA method. Analysis of the results of laboratory control of series of vaccines against hepatitis B using a biological method for immunogenicity evaluation based on ED50 analysis confirms persistently high immunogenic activity of the Russian commercial vaccines intended for prophylaxis of hepatitis B. The confirmed comparability of methods allows the number of in vivo tests to be further reduced in favor of the enzyme immunoassay authentically characterizing the produced drug.

scholarly journals Development and validation of a method for the determination of the specific activity of recombinant monoclonal antibody eculizumab

2020 ◽  
Vol 15 (2) ◽  
pp. 77-85
Author(s):  
D. I. Zybin ◽  
A. S. Seregin ◽  
A. D. Askretkov ◽  
N. V. Orlova ◽  
Yu. A. Seregin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Pharmaceutical Analysis ◽  
Comparative Evaluation ◽  
Specific Activity ◽  
In Vitro Method ◽  
Recombinant Monoclonal Antibody ◽  
Development And Validation ◽  
First Time

Objectives. Developing reliable and accurate analytical methods is necessary for comparative pharmaceutical analysis using physicochemical, biological (in vitro), preclinical, and clinical trials. The main objective of this study was to develop and validate an in vitro method for determining the specific activity of the recombinant monoclonal antibody eculizumab.Methods. The method of indirect enzyme immunoassay was used in the study.Results. A method for determining the specific activity of the humanized recombinant monoclonal antibody eculizumab was described and validated for the first time. A comparative evaluation of the specific activity of Soliris® (Alexion Pharmaceuticals Inc., USA), and its biosimilar PRK-001 (Pharmapark, Russia) was performed using the developed method.Conclusions. The similarity of PRK-001 and the original Soliris® in relation to their specific activity, that is, binding to the human complement system C5 protein, was proved. 

scholarly journals Assessment of the competitiveness of catering companies

2020 ◽  
Vol 23 (9) ◽  
pp. 77-85
Author(s):  
Nataliia Kyrnis
Keyword(s):  
High Tech ◽  
Internal Factors ◽  
Approach Method ◽  
Methodological Approaches ◽  
Determination Of Parameters ◽  
Post Industrial ◽  
Optimal Evaluation ◽  
Selection Of

Modernization of the Ukrainian economy under the influence of global post-industrial trends mainstreams the priority of development of science-intensive, high-tech, innovative activities. Modernization creates the preconditions for strengthening competitive positions in the national and global markets. In modern conditions, the issue of assessing the competitiveness of catering companies is relevant. High competitiveness of the enterprise is the main condition of its activity and development. The purpose of the study is to assess the competitiveness of catering companies in the market of catering services in Ukraine. The study proposes methodological approaches to assessing the competitiveness of catering companies. Methodological approaches include continuity of monitoring, determination of parameters, indicators and criteria, selection of optimal evaluation methods. In the process of research the following methods were used: sociological survey, simple ranking, integrated assessment and factor method, system approach method. It was established that methodical approaches to assessing the competitiveness of catering companies include justification and development of a system of socio-economic indicators and criteria (product quality, quality of service, quality and organizational staff, efficiency, efficiency of personnel management, price). Methodological approaches include a set of indicators that form an integrated indicator of competitiveness. This indicator allows to consider objective differences in the dynamics of enterprises, the conditions of use of available resources in the process of forecasting and development of enterprises. A comprehensive diagnosis was carried out based on an integrated indicator of the competitiveness of restaurants on special orders (catering), during which it was determined that its level has a significant differentiation according to the indicators of the studied enterprises, which are due to both external and internal factors, which allows the development of strategic priorities and specific measures to develop the image of the enterprise and increase its competitiveness.

scholarly journals Using Time Lapse Monitoring for Determination of Morphological Defect Frequency in Feline Embryos after in Vitro Fertilization (IVF)

Animals ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 3 ◽  
Author(s):  
Barbara Kij ◽  
Joanna Kochan ◽  
Agnieszka Nowak ◽  
Wojciech Niżański ◽  
Sylwia Prochowska ◽  
...  
Keyword(s):  
Time Lapse ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Vitro Fertilization ◽  
First Results ◽  
Morphological Defects ◽  
Defect Frequency ◽  
Selection Of

Some human, bovine, and mouse in vitro fertilized (IVF) embryos with morphokinetic abnormalities such as fragmentation, direct cleavage, and cytoplasmic vacuoles have the potential to reach the blastocyst stage, which is related to a high potential for implantation. The latest techniques of embryo development observation to enable the evaluation and selection of embryos are based on time lapse monitoring (TLM). The aim of this study was to determine the frequency of morphological defects in feline embryos, their competence to reach the blastocyst stage, and their ability to hatch. Oocyte-cumulus complexes were isolated after the scarification of ovaries and matured in vitro. Matured oocytes were fertilized in vitro by capacitated spermatozoa. Randomly selected oocytes were observed by TLM for seven-to-eight days. Out of 76 developed embryos, 41 were morphologically normal, of which 15 reached the blastocyst stage. Of 35 abnormally developed embryos, 17 reached the blastocyst stage, of which six had single aberrations and 11 had multiple aberrations. The hatching rate (%) was 15.6% in normally cleaving embryos, 6.25% in embryos with single aberrations, and 3.33% in those with multiple aberrations. The present study reports the first results, found by using TLM, about the frequency of the morphological defects of feline embryos, their competence to reach the blastocyst stage, and their ability to hatch.

Biochemical characterization a digestive trypsin in the midgut of large cabbage white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae)

2017 ◽  
Vol 108 (4) ◽  
pp. 501-509
Author(s):  
A. Sharifloo ◽  
A. Zibaee ◽  
J. Jalali Sendi ◽  
K. Talebi Jahroumi
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Pieris Brassicae ◽  
Larval Midgut ◽  
Posterior Midgut ◽  
Diethylaminoethyl Cellulose ◽  
Cabbage White Butterfly ◽  
Fast Flow

AbstractA comprehensive study on digestive trypsin was undertaken in the larval midgut of Pieris brassicae L. Results of enzymatic compartmentalization showed a significantly higher activity of crude trypsin in the anterior larval midgut rather than posterior-midgut. Using Diethylaminoethyl cellulose fast flow column chromatography a purified trypsin was obtained by specific activity of 21 U mg−1 protein, recovery of 22%, purification fold of 28-fold and molecular weight of 25 kDa. This purified enzyme showed the highest activity at pH 8 and the corresponding temperature of 40°C. However, the specific inhibitors used including 4-(2-Aminoethyl) benzenesulfonyl fluroride hydrochloride, N-p-Tosyl-L-lysine methyl ester hydrochloride and Soybean Trypsin Inhibitor significantly lowered the activity of the purified enzyme in vitro. Moreover, the activity of trypsin and likewise the nutritional indices were significantly altered in the larval midgut feeding upon the leaves treated by 1 mM concentration of each inhibitor in comparison with control. Determination of enzymatic characteristics of insect trypsins is crucial in paving the path for controlling pests by potential natural compounds via transgenic plants.

Determination of rumen microbial growth in vitro from32P-labelled phosphate incorporation

1977 ◽  
Vol 38 (1) ◽  
pp. 101-114 ◽  
Author(s):  
C. J. Van Nevel ◽  
D. I. Demeyer
Keyword(s):  
Microbial Growth ◽  
Specific Activity ◽  
Rumen Fluid ◽  
List Type ◽  
Substrate Protein ◽  
Isotope Method ◽  
Total Growth ◽  
N Incorporation

1.The extracellular phosphate pool in incubations of rumen fluid or washed cell suspensions of mixed rumen bacteria (WCS) was labelled with32P. From the constant extracellular phosphate pool specific activity and the amount of radioactivity incorporated during incubation, the amount of P incorporated in the microbial fraction was calculated. From the value for nitrogen: P determined in microbial matter, the amount of N incorporated was calculated as a measure of microbial growth.2.Incorporation of soluble non-protein-N in incubations devoid of substrate protein was 50 and 80 % of the values obtained using the isotope method for rumen fluid and WCS respectively. It is suggested that results obtained using the former method reflect 'net growth' of micro-organisms which is the result of simultaneous growth and degradation. The isotope method measures 'total growth', as isotope incorporation is not affected by degradation of non-growing cells.3.Incorporation of32P in P-containing microbial components (mainly nucleic acids) was compared with net synthesis of these components in incubations of WCS. The results showed different specific rates of synthesis and degradation for all components studied. It is concluded that the composition of microbial matter changed during growth.4.When N incorporation, calculated from results obtained using the isotope method in incubations with rumen fluid, was compared with the amount of carbohydrate substrate fermented and the type of fermentation, values between 18.3 and 44.6 g N incorporated/kg of organic matter fermented were obtained. Low values were associated with large proportions of the substrate being fermented to lactate and the use of glucose instead of disaccharides as substrate. Part of the variation could also be attributed to differences in incubation period, reflected in different proportions of polysaccharide formed.5.The use of isotopes for determination of rumen microbial growth in vitro is critically discussed.

Determination of 21-hydroxylase autoantibodies: inter-laboratory concordance in the Euradrenal International Serum Exchange Program

2015 ◽  
Vol 53 (11) ◽  
Author(s):  
Alberto Falorni ◽  
Vittorio Bini ◽  
Corrado Betterle ◽  
Annalisa Brozzetti ◽  
Luis Castaño ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Rater Agreement ◽  
Exchange Program ◽  
Area Under Roc Curve ◽  
Cutoff Levels ◽  
Commercial Kit ◽  
Selection Of

Abstract21-Hydroxylase autoantibodies (21OHAb) are markers of an adrenal autoimmune process that identifies individuals with autoimmune Addison’s disease (AAD). Quality and inter-laboratory agreement of various 21OHAb tests are incompletely known. The objective of the study was to determine inter-laboratory concordance for 21OHAb determinations.Sixty-nine sera from 51 patients with AAD and 51 sera from 51 healthy subjects were blindly coded by a randomization center and distributed to 14 laboratories that determined 21OHAb, either by an “in-house” assay (n=9) using in vitro-translatedIntra-assay coefficient of variation ranged from 2.6% to 5.3% for laboratories using the commercial kit and from 5.1% to 23% for laboratories using “in-house” assays. Diagnostic accuracy, expressed as area under ROC curve (AUC), varied from 0.625 to 0.947 with the commercial kit and from 0.562 to 0.978 with “in-house” methods. Cohen’s κ of inter-rater agreement was 0.603 among all 14 laboratories, 0.691 among “in-house” laboratories, and 0.502 among commercial kit users. Optimized cutoff levels, calculated on the basis of AUCs, increased the diagnostic accuracy of every laboratory (AUC >0.9 for 11/14 laboratories) and increased the Cohen’s κ of inter-rater agreement. Discrepancies in quantitation of 21OHAb levels among different laboratories increased with increasing autoantibody levels.The quality of 21OHAb analytical procedures is mainly influenced by selection of cutoff value and correct handling of assay materials. A standardization program is needed to identify common standard sera and common measuring units.

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