Иммуноферментные системы и набор реагентов для определения бацитрацина в пищевых продуктах

Two test-systems for a direct and an indirect enzyme-linked immunosorbent assay (ELISA) of peptide antibiotic bacitracin (BC) were developed and studied. For the both systems, polyclonal antibodies were obtained by immunizing rabbits with a conjugate of BC with keyhole limpet hemocyanine synthesized using reaction between the peptide and the high molecular weight protein in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The product of BC linking to thyroglobulin which was activated with EDC and N-hydroxysulfosuccinimide served as conjugated antigen on a solid phase in the indirect ELISA. For the direct ELISA, the antibodies against BC were immunochemically immobilized onto microplate surface, while the liquid phase contained a conjugate of BC with horseradish peroxidase. This conjugate was obtained by successive reactions of antibiotic amino groups coupling to periodate oxidized carbohydrate chains of enzyme and the reducting of formed Shiff’s base with sodium borohydride. Conjugated antigens binding to anti-BC antibodies provided maximum colorimetric signals of 2.0 and 1.2 optical units for the direct and indirect ELISA, respectively, and depended on BC content in the liquid phase. Antibiotic concentration that caused the inhibition of binding by 50 % was 2.6 ng/ml in the direct ELISA and 10.0 ng/ml in the indirect ELISA. The simple and sensitive direct ELISA system was used as a prototype of the finished reagent kit and a method for measurements with technical-analytical parameters and metrological characteristics allowing the determination of BC residues in a variety of foods including 14 items in a concentration range of 9.0 to 405.0 pg/kg with proper accuracy and precision.Разработаны тест-системы для непрямого и прямого иммуноферментного анализа (ИФА) пептидного антибиотика бацитрацина (Бц). Поликлональные антитела для обеих тест-систем получены иммунизацией кроликов конъюгатом Бц и гемоцианина улитки, который синтезировали взаимодействием пептида с белком в присутствии 1-этил-3-(3-диметиламинопропил)-карбодиимида (EDC). В системе непрямого ИФА твердофазным конъюгированным антигеном служил продукт присоединения Бц к тироглобулину, предварительно активированному EDC и N-гидроксисульфосукцинимидом. Для прямого ИФА на поверхности лунок микропланшета были иммунохимически иммобилизованы антитела к Бц, а жидкая фаза содержала конъюгат Бц с пероксидазой из корней хрена, полученный путем последовательных реакций присоединения аминогрупп Бц к окисленной периодатом углеводной части фермента и восстановления образовавшегося основания Шиффа борогидридом натрия. В системах прямого ИФА и непрямого ИФА связывание конъюгированных антигенов с антителами к Бц обусловливало максимальные колориметрические сигналы около 2,0 и 1,2 единиц оптической плотности соответственно и зависело от содержания Бц в жидкой фазе. При этом концентрации антибиотика, вызывающие ингибирование связывания на 50 %, составили 2,6 нг/мл в прямом ИФА и 10,0 нг/мл в непрямом ИФА. Простая и чувствительная система прямого ИФА послужила прототипом для создания готового набора реагентов и методики выполнения измерений, технико-аналитические параметры и метрологические характеристики которых позволяют определять остаточные количества Бц в пищевой продукции животного происхождения расширенного перечня, включающего 14 наименований, в диапазоне от 9,0 до 405,0 мкг/кг с надлежащей правильностью и точностью

Изучение фармакокинетического профиля лекарственного препарата Гидазепам®, таблетки, 50 мг в исследовании биоэквивалентности

Aim. The primary aim of this study was to evaluate the pharmacokinetic parameters and confirm the bioequivalence of drugs containing gidazepam, namely Gidazepam® (Valenta Pharm, Russia) and Gidazepam VIC (VIVA Pharm, Republic of Kazakhstan), after a single administration of 1 tablet (50 mg) to healthy volunteers under fasting conditions. The secondary aim was a comparative analysis of safety profiles (adverse events) after a single administration of the studied drugs.Materials and methods. An open, randomized, crossover, two-period comparative study of pharmacokinetics and bioequivalence with adaptive design was conducted in healthy volunteers. Blood sampling was performed 15 minutes before and 20 min, 40 min, 1 h, 2 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h after drug administration. High-performance liquid chromatography-tandem mass spectrometry was used for the evaluation of gidazepam and its metabolite (desalkylgidazepam) concentration with the subsequent calculation of pharmacokinetic parameters.Results. From both formulations, gidazepam was quickly absorbed and biotransformed into an active metabolite. Studied drugs had similar pharmacokinetic profiles, as 90% confidence intervals for the ratio of geometric means for Cmax and AUC(0-72) were within the bioequivalence acceptance range of 80.00–125.00 %. No adverse events were recorded as a result of clinical, laboratory or instrument evaluations during the study.Conclusion. Study drugs are considered bioequivalent and show comparable tolerability after a single administration under fasting conditions.Цель исследования. Изучение фармакокинетики и подтверждение биоэквивалентности препаратов, содержащих гидазепам: Гидазепам® (АО «Валента Фарм», Россия) и Гидазепам VIC (ТОО «ВИВА ФАРМ», Республика Казахстан) при однократном приёме 1 таблетки (50 мг) здоровыми добровольцами натощак. Дополнительной целью исследования являлся сравнительный анализ данных о нежелательных явлениях при однократном приёме изучаемых препаратов.Материал и методы. Проведено открытое, рандомизированное, перекрёстное, двухпериодное с адаптивным дизайном исследование сравнительной фармакокинетики и биоэквивалентности с участием здоровых добровольцев. Отбор образцов крови осуществляли за 15 мин до приёма и через 20 мин, 40 мин, 1 ч, 2 ч, 3 ч, 3,5 ч, 4 ч, 4,5 ч, 5 ч, 6 ч, 8 ч, 12 ч, 24 ч, 48 ч и 72 ч после приёма лекарственных препаратов. С помощью метода высокоэффективной жидкостной хроматографии с тандемной масс-спектрометрией определяли концентрацию гидазепама и его метаболита (дезалкилгидазепама) для последующего расчёта фармакокинетических показателей.Результаты исследования. Гидазепам в составе двух сравниваемых лекарственных препаратов характеризовался быстрой абсорбцией и биотрансформацией с образованием активного метаболита. Препараты исследования обладали эквивалентным фармакокинетическим профилем, поскольку 90 % доверительные интервалы для отношения геометрических средних величин параметров Cmax и AUC(0–72) не выходили за установленные пределы 80,00–125,00 %. По данным клиниколабораторного и инструментального наблюдения за время проведения исследования у добровольцев не было зарегистрировано ни одного нежелательного явления.Заключение. Сравниваемые препараты являются биоэквивалентными и демонстрируют одинаковый профиль переносимости при однократном приёме натощак

Real-time measurement of relative sensor position changes by ultrasonic signal evaluation

Ultrasonic testing is considered to be one of the most commonly applied nondestructive testing techniques for flaw detection and material characterization. Traditional Nondestructive Testing (NDT) provides detection of material discontinuities that may cause failure within the designed lifetime of a part or component. In addition, Quantitative Nondestructive Testing (QNDT) provides means to obtain required information about type, size and location of deficiencies to the integrity of the inspected structure and further use under specific, given load conditions. The "Acoustic Mouse" technique has been developed as a tool for manual ultrasonic inspection to provide test results that can be evaluated quantitatively. The ultrasonic data are processed by real-time variation methods to extract position information from backscattered acoustic noise and geometric scatter signals in the inspection volume. The position and positional changes of the "Acoustic Mouse" sensor (transducer) are determined by the sequential analysis of ultrasonic data (highresolution sector-scans), which are acquired and reconstructed using the Sampling Phased Array technique. The results of first experiments conducted with linear scanning and intentional lift-offs demonstrate sufficient accuracy in position measurements

Models of dependence of the quantity of the <i>Streptococcus pneumoniae biomass</i> and his capsular polysaccharide from the composition of the feeding environment

Aim.The development of a semi-synthetic nutrient medium that provides the maximum amount of capsular polysaccharide (CPS). Materials and methods. We used the strain 521 of S. pneumoniae serotype 23F. Cultivation was carried out in test tubes with 10 ml of polysynthetic nutrient medium of a specific composition. The amount of polysaccharide in the samples was determined using rocket immunoelectrophoresis. Building models and comparing the effects of various components was carried out according to the methodology specified in the tutorial. The calculation of the coefficients of the equation and the assessment of the adequacy of the equations themselves was carried out using RStudio version 1.0.153. Results. As a result of a series of experiments, the coefficients of the regression equations were calculated, their significance was evaluated, and models of dependence of biomass production and CPS were constructed depending on the composition of the nutrient medium. To solve the problem, an experiment was carried out according to the Box-Wilson method. The peptone and glucose concentrations were selected as optimized parameters. The step size ΔSi in the increasing gradient direction was calculated based on the coefficients of the regression equation. At the same time, the exact nature of the dependence was determined. The optimal calculated concentrations of peptone and glucose, at which the formation of CPS is maximum, are 32.6 and 12.1 g/l, respectively. In this case, the forecast yield of the polysaccharide is 239 mg/l. Conclusion. Using the method of fractional factorial experiment, models of the dependence of the biomass amount of S. pneumoniae and its capsular polysaccharide on the composition of the nutrient medium were obtained. The optimal concentrations of the components of the medium were found, which make it possible to increase the level of biomass formation by 10% compared to the standard formulas, and the CPS — by 1.5—2 times

Вплив лізату клітин кісткового мозку на біохімічні показники крові мишей та розвиток аденокарциноми Ерліха

The paper presents the results of investigation of influence of bone marrow cells lysate from mouse (BMCm) on blood biochemistry and development of tumor on transplantable Ehrlich carcinoma model in Balb2 mices. As a result, it was found that injection of BMCm lysate inhibited growth of a solid form of Ehrlich carcinoma in the thigh muscle of experimental animals by 16 % after a single dose, and at 31 % at the multiple procedures. It has been found that the infusion of stem cells mass decreased rate of tumor growth by 37 %. The tumor has not occurred in 10 % of animals. In addition, the injection of BMCm lysate contributed normalization of some indicators of blood chemistry test of animals. Thus, the total protein increased only in 15-18 % after a single dose of BMCm lysate injection and remained normal during multiple procedures. Albumin level decreased by 29,5 % after a single injection of BMCm lysate. When repeated, this procedure led to normalization of albumin level. ALT activity in a single BMCm lysate injection group increased to 103-137 %, while in multiple injections group ALT activity was only by 48-73 % higher than in control samples. Confirmation of normalization of thigh tissue structure was also obtained in histological observation. These data demonstrated the antitumor effect of inactivated lysate of BMCm on the Ehrlich carcinoma model.В работе представлены результаты исследования влияния лизата клеток костного мозга мыши (ККМ) на показатели биохимии сыворотки крови и развитие опухолевого процесса в организме мышей линии Balb2 на модели перевивной карциномы Эрлиха. В результате было обнаружено, что введение лизата ККМ способствовало торможению развития солидной формы карциномы Эрлиха в мышце бедра подопытных животных на 16 % при однократном введении, и на 31 % при многократном введении. Обнаружено, что введение лизата ККМ снижало скорость роста опухолей на 37 %. Опухоль не развилась у 10 % животных. Кроме того, введение лизата ККМ мышей способствовал нормализации некоторых показателей биохимии крови подопытных животных. Так, общий белок повышался только на 15-18 % при однократном введении лизата ККМ, и оставался в норме при многократном введении. Уровень альбумина при однократном введении лизата ККМ снижался на 29,5 %. При многократном введении этот показатель был в пределах нормы. Активность АЛТ при однократном введении лизата повышалась на 103-137 %, а при многократном введении только на 48-73 %, по сравнению с контролем. Подтверждение нормализации состояния мышечных тканей бедра подопытных животных получено также на образцах гистологических препаратов. Приведенные данные демонстрируют противоопухолевый эффект от введения инактивированной суспензии стволовых клеток на модели карциномы Эрлиха.У роботі наведені результати дослідження впливу лізату клітин кісткового мозку миші (ККМм) на біохімічні показники сироватки крові та розвиток пухлинного процесу в організмі мишей лінії Balb2 на моделі перевивної карциноми Ерліха. У результаті виявлено, що введення лізату ККМ сприяло гальмуванню розвитку солідної форми карциноми Ерліха у м’язі стегна піддослідних тварин на 16 % при одноразовому введенні, та на 31 % при багаторазовому введенні. Виявлено, що введення лізату ККМ знижувало швидкість росту пухлин на 37 %. Пухлина не розвинулась у 10 % тварин. Крім того, ін'єкція лізату ККМ мишей сприяла нормалізації деяких показників біохімії крові піддослідних тварин. Так, загальний білок підвищується лише на 15-18 % при одноразовому введенні лізату та залишався в нормі при багаторазовому введенні. Рівень альбуміну при однократному введенні лізату знижується на 29,5 %. При багаторазовому введенні цей показник був у межах норми. Активність АЛТ при однократному введенні лізату підвищується на 103-137 %, а при багаторазовому введенні лише на 48-73 %, порівняно з контролем. Підтвердження нормалізації стану м’язових тканин стегна піддослідних тварин отримано на зразках гістологічних препаратів. Наведені дані демонструють протипухлинний ефект від уведення інактивованої суспензії ККМ на моделі карциноми Ерліха

A case of generalized tuberculosis in a school-age child with late detected HIV infection

The paper describes a case of generalized tuberculosis in a HIV-infected child. The specific feature of the case is the late detection of both tuberculosis and HIV infection. Tuberculosis was diagnosed on the basis of the data of multislice spiral computed tomography and bacteriological examination of bronchial washes. The case demonstrates the development of severe generalized tuberculosis in the child with untimely detected HIV infection, as well as pediatricians’ low alertness to HIV infection in children and inadequate HIV detection works among pregnant women

Experimental Protein-Containing Preparations <i>Streptococcus pneumoniae</i>, Obtained from Fresh Isolated Strains and Museum

Relevance. Vaccines based on capsular polysaccharides of pneumococci are not active against serotypes that are not included in the vaccine, non-capsulated strains and do not protect against carriage caused by other serotypes. Their use leads to the replacement of dominant serotypes of pneumococci, the appearance of highly virulent strains, changes in the microbial landscape of mucous membranes due to the appearance of other etiologically significant pathogens of respiratory tract diseases. This requires the creation of intraspecific anti-pneumococcal immunity, which will be facilitated by the development of serotype-dependent drugs, which will include protein-containing antigens of pneumococci.Objective. Study of serotype-independent activity of protein-containing antigenic components obtained from freshly isolated and archival strains of S. pneumoniae.Materials and methods. Strains of three serotypes of S. pneumoniae were used: archival strains of serotypes 6B N296, 19F N 298 and 10A N297 and freshly isolated serotypes 6B N 1121, 19F N 1055 and serogroup 10 N 1193 (from the cerebrospinal fluid of patients with purulent meningitis).. In the experimental protein-containing preparations EPCP obtained, the protein content was determined. Protective activity of EPCP and virulence of strains were determined in the model of intraperitoneal immunization and infection of BALB/c mice; LD50 was calculated using the generally accepted modified Kerber formula. The immunophenotype of lymphocytes previously isolated from donor-mice whole blood was studied by flow cytometry. Statistical analysis of the materials was carried out using parametric and nonparametric methods using the application package «Statistica for Windows», ver. 7.0 (Stat Soft, Inc); in statistical analysis, the significance level of p was assumed to be &lt; 0.05.Results and discussion. During cultivation there was the analysis of growth dynamics showed intensive accumulation of biomass of the archival strain N296 with low virulence, and lower - by virulent strain N 1121 isolated from the patient's cerebrospinal fluid. The protein content of drugs from serotype 6B strains did not differ. The strains of other serotypes isolated during the generalized infectious process were also more virulent than the archival strain. The effect on the immunophenotype of human lymphocytes of fractions of 50-100 kDa isolated from the initial preparations obtained by culturing serotype 6B, significantly increased only under the influence of the preparation from the archival strain. In the study of protective activity of the initial preparations from strains of serotype 6B and fractions with MM 50-100 kDa only triple immunization with the initial preparation from strain N 296, at a dose of 20 micrograms of protein per mouse, led to significantly greater survival of immunized mice from infection with the newly isolated virulent strain N 1121 of homologous serotype. A fraction of 30-100 kDa provided protection of mice twice immunized with 50 mkg of protein per mouse, with a high efficacy index of 8.9, even after infection with a freshly isolated strain of heterologous S. pneumoniae serotype 3 N 10196.Conclusion. The protein-containing fraction with MM 30-100 kDa obtained from a low virulent archival strain of S. pneumoniae serotype 6B N296 possessed protective activity against a newly isolated virulent strain of heterologous serotype after double immunization. Under the action of the studied protein-containing fractions, activation of the cellular component of immune system with the involvement of innate immunity effectors and T-lymphocytes is shown. These data can be considered as a evidence for further study of EPCP to assess the possibility of their use in the design of anti-pneumococcal drug with serotype-independent protective activity

Immunogenic Activity of Secreted Protein-Based Compounds Staphylococcus aureus № 6

Difficulties in the therapy of infection, caused by S. aureus, depends on the resistance of staphylococci to antibiotics, proceeding to chronicity of the diseases and the development on that background the depression of innate immunity functions and decreasing of the host resistance to the infection. In spite of tremendous efforts of researchers, up to date there are no commercial antistaphylococcal vaccine the efficacy of which would be proved in the completed clinical trials.Aim: obtaining secreted protein-based substances of the S. aureus № 6 and investigation their immunogenicity.Material and methods. Secreted protein-based substances (SPS) were obtain as: «initial» – from the filtrates of the culture fluid of the S. aureus № 6, grown to the end of the exponential phase according to the technology described previously [9], and I SPS and II SPS – after the ion-exchange chromatography of the «initial» SPS on the columns with Q-Sepharose and DEAE-Sepharose. The level of specific IgG antibodies in sera of immunized rabbits and mice determined in ELISA, the immunogenic activity evaluated in experiments of active and passive protection from the challenge performed on BALB/с mice and also by the determination of the bacterial content in organs and in the test of abscesses formation in kidneys. Results. Investigated SBS possessed the antigenic activity (the level of specific IgG antibodies in sera of immunized animals increased 2.2 – 7.5 times compared to the control groups), that is favor of the activation of the adaptive immunity system and significant protective activity revealed in experiments of active (index of efficacy 2.63 – 4.28) and passive protection. The immunization of mice with the «initial» and II SPS led to significant decrease of the number of colonyforming units of S. aureus and formation of abscesses in kidneys of mice. It is evidently, that investigated SPS, influence on the severity of staphylococcal infection and possesses the therapeutic effect.Conclusion. The preformed complex analysis at the current stage allowed to reveal perspectives of the further study of «initial» and II SPS in pre-clinical trials, as candidates, possessing the high protective facilities, for including them in the drug composition for immune prophylaxis and immunotherapy of diseases, caused by S. aureus