Validation procedure for method of monitoring the biological activity of reсombinant human interleu-
e x p e r i m e n t a l w o r k s e x p e r i m e n t a l w o r k s UDC 573.6.083.3+573.6.086.8 CharaCteristiCs of enzyme-linked immunosorbent assay for deteCtion of igg antibodies speCifiC to Сhlamydia trachomatis heat shoCk protein (hsp-60) O. Yu. GalkIN, a. B. BeSaraB, T. N. lUTSeNkO National registered). In 4 out of 15 intralaboratory panel serum samples initially identified as nega tive, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive. The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples. Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result. High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven. Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant elISa-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents. K e y w o r d s: enzyme-linked immunosorbent assay, Chlamydia trachomatis, heat shock protein, anti-idiotypic antibodies, sensitivity, specificity.C hlamydial genitourinary infection (GUI) is one of the most common sexually transmitted infections. According to World Health Organization, Chlamydia trachomatis infects about 90 million people each year by sexual transmission. In Ukraine, the rate of chlamydial GUI is 80 cases per 100,000 population. Nearly 16% of pregnant women are infected with C. trachomatis. About 50-60% of tubular infertility cases are caused by chlamydial infection. A quarter of all ophthalmic and respiratory diseases in newborn and younger children are associated with chlamydial infection [1,2].Effective diagnosis is one of important components of chlamydial GUI control, which can be realized through both direct (detection of antigens, nucleic acids, pathogenic agent microscopic examination and cultivation) and indirect (specific antibo dies detection) techniques. One of the commonly used methods in diagnostics is enzyme-linked immunosorbent assay (ELISA) that allows differential diagnosis -determination of disease stage and course, which is especially important in chronic conditions. For that purpose blood serum (plasma) and human biological fluids are tested for the presence of IgM, IgA and IgG classes of specific antibodies to pathogen's antigens [2,3].Prolonged persistency of C. trachomatis increases the expression of heat shock protein with molecular mass 60 kDa (HSP-60), which is highly
The purpose of the work was to rise of biosynthesis of the human recombinant interleukin-7 (rIL-7) in E. coli by optimizing the quantitative and qualitative composition of the culture medium. As a result of optimization of the quantitative composition of organic and mineral components of the culture medium using the matrix of planning a fractional factor experiment, it was possible to increase the synthesis of rIL-7 by 1.3 times. It was proved that the use of supplements to the culture medium in the form of extracts of Clivia miniata and Zephyranthes grandiflora (0.5-1.0%) stimulated the rIL-7 synthesis in recombinant bacterial cells in 1.3-1.4 times. The use of phytoextracts at a concentration of 5.0% and more caused an inhibitory effect on the producer biosynthetic properties. It has been established that the modulation of the biosynthetic activity of recombinant bacteria is due to the interaction of the biologically active substances of plant extracts with the surface structures of the bacterial cell, namely, porins (OmpC and OmpF proteins). The stimulating effect of vitamin K1 (15-25 mg per 1 ml of culture medium) in the liposomal form was proved; such approach allowed increasing the synthesis of rIL-7 in 1.34-1.39 times. And the use of liposomal form of plant extracts as an approach to increasing the bioavailability of biologically active substances is not appropriate.
The article presents the results of the comparative development of different variants of immunoaffinity sorbents for the purification of human IL-7, namely: Sepharose 6B and sol-gel materials based on tetraethoxysilane (TEOS). The optimal conditions for the synthesis of a chromatographic sorbent based on TEOS, which provide acceptable characteristics of the immobilization of a biological substance, were experimentally established. The sorbent based on TEOS and Sepharose 6B provide identical characteristics for the sorption of antibodies after 12 hours of the experiment. To establish rational conditions for immunoaffinity chromatography, various combinations of sorbents, anti-IL-7 monoclonal antibodies, and elution solutions were studied. It was shown that a variant of immunoaffinity chromatography based on TEOS sorbent using 325D2 anti-IL-7 monoclonal antibodies and 4M magnesium chloride solution as eluate, is more effective. Immunochromatographic columns synthesized on the basis of Sepharose 6B and TEOS are characterized by high recovery rates (at least 95%) in a wide range of human IL-7 concentrations (0.05...20 μg/ml), however, the column based on Sepharose 6B is less than stable with repeated use (more than 12 cycles without regeneration). The developed method makes it possible to obtain recombinant human IL-7 with a purity of 98%.
Background. Quality management is a very important activity of any industrial enterprise, especially when it comes to manufacturing products in health care (drugs, including medical immunobiological preparations, medical devices, cosmetics, dietary supplements, etc.). Risk-oriented thinking enables an organization to identify factors that may cause rejection of its processes and its quality management system for planned results to establish preventive controls to minimize negative impacts and the greatest possible shaft advantage of the opportunities as they arise. The logical continuation of the risk assessment is validation processes. This article is a fragment of the complex scientific and technical work on the development and standardization of biotechnology products based on recombinant human interleukin-7 (rhIL-7). Objective. Justification for approaches to risk assessment in the technology for getting preparation based on rhIL-7 in the form of nasal spray and particularly risk assessment of the production process, and perspective validation of developed technologies. Methods. The methodology of risk assessment according to ISO 14971:2009 "Medical devices. Guidelines for risk management" was used. Pharmacopoeial methods were used to control the process and product quality (potentiometric determination of pH, osmolality, sterility). The integrity of the filters was determined by the method according to the manometer producer. Results. The work consistently study approaches to risk assessment in the technology for getting preparation based on rhIL-7 in the form of nasal spray and particularly risk assessment of the production process, and the validation of promising technologies developed. Selection of guidelines on risk assessment was conducted for Guidelines for Good Manufacturing Practices, ISO 9001:2015, ISO 13485:2005, and ISO 14971:2009. The greater acceptability of the use of the past two standards was showed. Technological production scheme is presented and qualitative approach to analyze the identified risks is applied. For the most critical stage of production (preparation of sterile solution) the validation process is conducted. Conclusions. The feasibility of risk assessment in the technology for getting preparation based on rhIL-7 in the form of nasal spray according to ISO 14971:2009 together with the system using risk analysis and critical control points is theoretically justified. It was found that the most critical step in the production is part of the preparation of a sterile solution, which includes the preparation of intermediate filtering and sterilizing drug. The conducted prospective validation of the most critical stages of the technology demonstrated its stability and compliance with established eligibility criteria.
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