Assessment of the Application of Erythrocytal Diagnosticum (Lyophilizate) in Detecting Tularemia Agent in Natural Foci

Tularemia is a zoonotic disease with a wide geographical dissemination, and its causative agent Francisella tularensis can be used as a bioterrorism agent. The aim of the study was to evaluate the use of a set of reagents “Erythrocytic immunoglobulin dry tularemia diagnosticum” (“DET-Ig”) with the help of control test strains and field material from natural tularemia foci. Materials and methods. Using the introduced erythrocyte diagnosticum, we studied the decontaminated cultures of test strains (F. tularensis Miura, F. tularensis 55, F. tularensis Schu, F. tularensis 15 NIIEG, Brucella abortus 544, B. melitensis 16-M, B. suis 1330, and Yersinia enterocolitica 64, Y. enterocolitica 178, Y. enterocolitica 383) and environmental samples suspected of containing F. tularensis. Results and discussion. It has been proven that the developed diagnosticum is specific, sensitive, and easy to use for routine diagnostics of tularemia. In the course of laboratory tests of the experimental series of the DET-Ig reagent kit, the possibility of qualitative determination of the tularemia agent in bacterial cultures, biological material and environmental samples in the reaction of indirect hemagglutination was demonstrated. Comparison of the results of use of erythrocyte diagnosticum in liquid and lyophilized forms showed the advantages of drugs after lyophilization: the possibility of transportation and long-term storage at any temperature conditions in various climatic conditions; the setting of the reaction is possible without the use of special diluents. The guaranteed storage term is set for two years (observation period). The results obtained indicate the prospects of introducing the developed drug into healthcare practice

Validation of Technological Process of Production of Liquid Brucellosis Diagnosticum for Agglutination Reaction, Suspension for Diagnostic Purposes

Presented are the results of validation of technological process of production of brucellosis diagnosticu

Development of a protective lyophilisation medium and conditions to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin

Liquid erythrocyte diagnostic preparations have a practical disadvantage; i.e., long-distance transportation involving possible non-compliance with cold-chain requirements may result in a complete loss of biological activity. A lyophilisation technology is necessary to ensure that the preparations retain their original properties for a long time. The aim of the work was to develop a protective medium and conditions for lyophilisation to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin. Materials and methods: Gelatin, thiourea, trehalose, sucrose, dextran, and Tween 80 were used as excipients for protective media. The authors used nine strains of homologous and heterologous microorganisms of different genera and species to control the lyophilised diagnostic preparation sensitivity and specificity. Evaluation of the main stability-related quality attributes (appearance of the dried preparation, loss on drying, solubility, appearance after reconstitution, appearance after settling, sensitivity, specificity) considered the temperatures specific to the climatic zones where the in vitro diagnostics is intended to be marketed and used. Results: The authors developed protective stabilising media with different compositions, used them in freeze-drying of the preparation and carried out control testing. The most promising was the lyophilisation medium containing a smaller amount of ingredients —6% of dextran, 0.06% of Tween 80 and up to 0.01% of sodium azide—as it was the simplest one to prepare and ensured complete preservation of the quality attributes. The authors carried out practical evaluation of lyophilisation procedures, and the 12–14-hour procedure proved to be the most cost-effective. Conclusions: The results of long-term, or real time, and accelerated stability testing of the lyophilised diagnostic preparation demonstrated the possibility of two-year storage at a labelled temperature of 2–8 °C, as well as at elevated and low temperatures of 30±2 °С and –18 °С, respectively. The tests showed no negative effects of the temperatures on the controlled quality attributes

Assessment of the Effectiveness of Using Magnoimmunosorbents for the Selective Concentration of Anthrax Agent Spores

The aim of the study was to assess the effectiveness of the developed anthrax magnoimmunosorbents (MIS) for the selective concentration of Bacillus anthracis spores and to increase the sensitivity of anthrax agent detection techniques, including when testing soil samples.Materials and methods. We used 10 vaccine strains of B. anthracis and 30 strains of closely related bacilli of the genus Bacillus (B. cereus – 15, B. thuringiensis – 10, B. megaterium – 5) with typical species properties. The work was performed on three experimental batches of magnoimmunosorbents. DNA extraction and PCR setting was carried out in compliance with the instructions for reagent panel for B. anthracis DNA detection “ApliSens Bacillus anthracis-FRT”.Results and discussion. It is shown that when using MIS, the sensitivity of the cultural method is increased by at least 7 times (taking into account the possibility of sorption of 1–10 or more spores on a sorbent particle). The sensitivity of the PCR method is improved by 10 times and amounts to 50 B. anthracis spores per 1 ml for the samples concentrated with the help of MIS. The sensitivity of the bacteriological method using MIS increases by a factor of 7.5 when testing the artificially contaminated with B. anthracis soil samples. Hence, application of the developed MIS makes it possible to significantly enhance the sensitivity of anthrax agent detection methods and can be considered as an effective means of sample preparation for the investigation of environmental objects (soil)

Experimental Peroxidase Conjugate for Detection of Specific Antibodies to Anthrax Agent in Enzyme Immunoassay

Anthrax poses a pressing issue for veterinary medicine and public health in many countries, including the Russian Federation, which necessitates the improvement and development of new, sensitive and specific diagnostic tools.The aim of the work was to create an experimental peroxidase conjugate for the detection of specific antibodies to the anthrax pathogen and to optimize the conditions for performing enzyme immunoassay (ELISA).Materials and methods. The peroxidase conjugate was constructed using horseradish peroxidase and Staphylococcus aureus protein A (Sigma-Aldrich, USA). Bacterial antigens isolated from strains of Bacillus anthracis 55ΔTPA-1Spo, B. anthracis Sterne 34 F2 were used as sensitizing agents. The developed experimental batches of the conjugate were tested in ELISA for the ability to bind antibodies in the blood sera of anthrax patients and vaccinated individuals. The sensitivity, specificity, and accuracy of the method were calculated using the built-in functions of the ROCR software package.Results and discussion. The peroxidase conjugate to detect specific antibodies to the anthrax pathogen in the study of clinical material has been developed; conditions for the ELISA performance have been optimized. To interpret the results of the study, a threshold value of the positivity coefficient was used, below which the result was considered negative, and at an equal or higher value, positive. The test demonstrated significant differences in the “positivity coefficient” indicator for the “Healthy”/“Sick” and “Healthy”/“Vaccinated” groups, while the differences between the “Sick”/“Vaccinated” groups were statistically insignificant. The maximum accuracy of the method was observed at blood serum dilutions of 1:250 and 1:500. 100 % intra-run, run-to-run and series-to-series reproducibility has been established for all positive samples. The sensitivity and specificity of the experimental peroxidase conjugates were 100 and 95.8 %, respectively, and the accuracy was 97.6 %

Научно-методические разработки биотехнологий производства иммунобиологических препаратов для экспресс-диагностики инфекционных заболеваний и детекции их возбудителе

The present article describes the scientific and methodological development of biotechnological manufacture of test-system components (diagnostic preparations) for instant diagnosis of plague, brucellosis, tularemia, anthrax, cholera. In this regard, in the first place the effective methods for obtaining complete antigenic complexes used for immunizing animals for the purpose of developing highly potent immune sera have been established. These antisera were used in determining optimum parameters of manufacture on the basis of their diagnosticums. Methodical basis of developing magnetic immunosorbents for selective concentration of infectious agents and their instant diagnosis methods has been mentioned. Moreover, the article describes the development of piezoelectric quartz crystal biosensors to detect plague, brucellosis and tularemia pathogens by gravimetric flow injection analysis, allowing to quickly implement the process of reliable identification of a test pathogen in antigen-antibody complex.Представлены научно-методические разработки биотехнологий производства компонентов тест-систем (диагностических препаратов) для экспресс-диагностики чумы, бруцеллеза, туляремии, сибирской язвы, холеры. Для этого, прежде всего, были отработаны эффективные методики получения полноценных антигенных комплексов, применяемых для иммунизации животных в целях получения высокоактивных иммунных сывороток. Полученные иммунные сыворотки были использованы при определении оптимальных параметров производства на их основе диагностикумов. Приведены методические основы конструирования магноиммуносорбентов для селективного концентрирования возбудителей инфекционных заболеваний и их индикации экспресс-методами. Кроме того, приведена разработка пьезокварцевых биосенсорных устройств для выявления возбудителей чумы, бруцеллеза, туляремии в гравиметрическом проточно-инжекционном анализе, позволяющих быстро реализовать процесс достоверного распознавания исследуемого патогена в образовавшемся комплексе антиген-антител

Разработка защитной среды высушивания и режима лиофилизации для стабилизации диагностикума эритроцитарного туляремийного иммуноглобулинового

Liquid erythrocyte diagnostic preparations have a practical disadvantage; i.e., long-distance transportation involving possible non-compliance with cold-chain requirements may result in a complete loss of biological activity. A lyophilisation technology is necessary to ensure that the preparations retain their original properties for a long time. The aim of the work was to develop a protective medium and conditions for lyophilisation to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin. Materials and methods: Gelatin, thiourea, trehalose, sucrose, dextran, and Tween 80 were used as excipients for protective media. The authors used nine strains of homologous and heterologous microorganisms of different genera and species to control the lyophilised diagnostic preparation sensitivity and specificity. Evaluation of the main stability-related quality attributes (appearance of the dried preparation, loss on drying, solubility, appearance after reconstitution, appearance after settling, sensitivity, specificity) considered the temperatures specific to the climatic zones where the in vitro diagnostics is intended to be marketed and used. Results: The authors developed protective stabilising media with different compositions, used them in freeze-drying of the preparation and carried out control testing. The most promising was the lyophilisation medium containing a smaller amount of ingredients —6% of dextran, 0.06% of Tween 80 and up to 0.01% of sodium azide—as it was the simplest one to prepare and ensured complete preservation of the quality attributes. The authors carried out practical evaluation of lyophilisation procedures, and the 12–14-hour procedure proved to be the most cost-effective. Conclusions: The results of long-term, or real time, and accelerated stability testing of the lyophilised diagnostic preparation demonstrated the possibility of two-year storage at a labelled temperature of 2–8 °C, as well as at elevated and low temperatures of 30±2 °С and –18 °С, respectively. The tests showed no negative effects of the temperatures on the controlled quality attributes.Практическое применение эритроцитарных диагностических препаратов выявило недостатки, связанные с их транспортировкой на значительные расстояния с возможным несоблюдением режимов холодовой цепи, что может привести к полной потере их биологической активности. Для стабилизации основных свойств жидких форм эритроцитарных диагностических препаратов в настоящее время необходима разработка технологии получения лиофилизированных форм диагностикумов, которая позволит сохранить первоначальные свойства препарата в течение длительного времени. Цель работы: разработка защитной среды высушивания и режима лиофилизации для стабилизации диагностикума эритроцитарного туляремийного иммуноглобулинового. Материалы и методы: были использованы вспомогательные вещества для подготовки защитных сред (желатин, тиомочевина, трегалоза, сахароза, декстран, твин 80). Для контроля чувствительности и специфичности лиофилизированных диагностикумов использовали 9 штаммов гомологичных и гетерологичных микроорганизмов разных родов и видов. При изучении стабильности основных показателей качества препаратов для диагностики in vitro (внешний вид высушенного препарата; потеря в массе при высушивании; растворимость, внешний вид восстановленного препарата; внешний вид препарата после отстаивания; чувствительность; специфичность) учитывали температуры различных климатических зон, в которых предполагается их реализация и использование. Результаты: разработаны и использованы стабилизирующие защитные среды с различным составом, с последующей сублимационной сушкой препарата и постановкой контрольных исследований. Наиболее перспективной признана среда высушивания для эритроцитарных диагностикумов, содержащая в своем составе меньшее количество ингредиентов — 6% декстрана, 0,06% твин 80 и азид натрия до 0,01%, как наиболее простая в исполнении и обеспечивающая полное сохранение качественных показателей препарата. Отработан рентабельный 12–14-часовой режим лиофилизации. Выводы: по совокупности полученных результатов изучения стабильности в реальном времени (долговременная стабильность) и ускоренном исследовании стабильности лиофилизированных форм диагностикума показана возможность хранения в течение двух лет при регламентированной температуре от 2 до 8 °С, а также в условиях повышенных и пониженных температур при 30±2 °С и минус 18 °С соответственно. Отрицательного влияния указанных температур на результаты контролируемых показателей не выявлено

Serological methods for detection of the causative agent of tularemia and their evaluation

Aim. A comparative study of serological methods for the detection of the causative agent of tularemia and their evaluation. Materials and methods. We used experimental diagnostic kits and test systems for the production of serological methods: indirect hemagglutination reaction (RGA); the reaction immunofluorescence (RIF); enzyme immunoassay (ELISA) using traditional microplate; IFA after selective concentration of the pathogen of tularemia in magnoimmunosorbents (MIS); microgravimetric analysis (MGA) based on piezoresistors (SP) and surface plasmon resonance (SPR). The experiments were carried out with homologous strains of tularemia microbe (test strains) and with strains of heterologous microorganisms in model experiments on tap water contaminated with different concentrations of the pathogen. Results. The parameters of each diagnostic method are determined and evaluated according to the following indicators: sensitivity (when working with pure cultures (test strains), contaminated samples of large volumes), specificity, time of setting and taking into account the results, informativeness, determining the modes of setting and accounting. Conclusion. The above diagnostic methods have their advantages and disadvantages. Therefore, when choosing a method, the researcher should be guided by the goals pursued. So, for screening studies it is advisable to carry out the formulation of ELISA, RIF, RGA, in identifying the pathogen in large volumes and contaminated samples, the effective use of selective concentration on MIS followed by the formulation of ELISA, to identify small amounts of samples and take into account the reaction in real time, it is possible to use MGA and SPR

Development of New Approaches of Obtaining the Hyperimmune Sera for Production of Medical Immunobiological Preparations

Developed were the new immunization schedules of obtaining plague, brucellar, anthrax, tularemia, cholera, leptospirosis, legionellosis and campilobacteriosis hyperimmune sera based on optimal combinations of specific protein antigenic complexes with immunomodulators, providing high specific immune response in 100 % of animals, significant reduction of immunization terms, material and labor input. Immune sera obtained are high quality biological material to be used for production of different diagnostic immunobiological preparations

Scientific and methodical development of biotechnological production of immunobiological preparations for instant diagnosis of infectious diseases and detection of pathogens

The present article describes the scientific and methodological development of biotechnological manufacture of test-system components (diagnostic preparations) for instant diagnosis of plague, brucellosis, tularemia, anthrax, cholera. In this regard, in the first place the effective methods for obtaining complete antigenic complexes used for immunizing animals for the purpose of developing highly potent immune sera have been established. These antisera were used in determining optimum parameters of manufacture on the basis of their diagnosticums. Methodical basis of developing magnetic immunosorbents for selective concentration of infectious agents and their instant diagnosis methods has been mentioned. Moreover, the article describes the development of piezoelectric quartz crystal biosensors to detect plague, brucellosis and tularemia pathogens by gravimetric flow injection analysis, allowing to quickly implement the process of reliable identification of a test pathogen in antigen-antibody complex