Eurasian Journal of Applied Biotechnology

THERAPY WITH CHIMERIC ANTIGEN RECEPTOR T-CELLS. OVERVIEW OF CLINICAL EFFICACY AND PROSPECTS FOR IMPLEMENTATION IN KAZAKHSTAN

2026-06-09T14:35:02+05:00

Chimeric Antigen Receptor T-cell therapy (CAR-T) is a form of adoptive cellular immunotherapy.

This review aims to inform healthcare professionals - including regulatory experts in gene and cell therapy - about the principles of CAR-T therapy. It discusses the mechanisms of action, provides an overview of currently approved CAR-T products, and summarizes up-to-date clinical efficacy data. The article also outlines the current status of implementing CAR-T in Kazakhstan. Key technological and organizational factors are described which determine the prospect for the localization of CAR-T manufacturing and the launch of the clinical use in the country.

The use of CAR-T therapy is expanding around the world, but this therapy is not implemented in Kazakhstan.

Hallmarks of CAR-T technology include that CAR-T products are manufactured individually for patients and consist of genetically modified T-lymphocytes. Currently, CAR-T therapy is approved and recommended for the treatment of B-cell leukemia, lymphoma, and multiple myeloma, and the therapy is particularly efficient in patients with relapsed or refractory disease which do not respond to standard therapies.

Findings from the literature review highlight the strong potential of CAR-T therapy to improve treatment outcomes for patients with refractory forms of leukemia and lymphoma in Kazakhstan’s oncology centers.

DISTRIBUTION OF KRAS/BRAF GENE MUTATIONS IN METASTATIC COLORECTAL CANCER IN KAZAKHSTAN PATIENTS

2026-05-20T16:34:12+05:00

Colorectal cancer (CRC) is characterized by significant molecular heterogeneity, with mutations in the KRAS and BRAF genes playing a key role in carcinogenesis and determining the effectiveness of anti-EGFR therapy. Data on the frequency of these mutations remain limited in the Kazakhstani population.

The aim of our study was to determine the frequency and spectrum of KRAS and BRAF mutations in primary tumors and metastatic lesions in patients with metastatic CRC using ddPCR and targeted sequencing.

The study included FFPE tumor tissue and liver metastasis samples from 30 patients. Screening for KRAS and BRAF mutations was performed using droplet digital PCR, and targeted NGS was used for validation and extended analysis of the mutational profile.

KRAS mutations were detected in 31.6% of primary tumors and 23.1% of metastases using targeted sequencing. The most common mutation was G12V, found in 21.1% of primary tumors and 15.4% of metastases. A greater diversity of KRAS variants was observed in primary tumors. BRAF mutations were detected significantly less frequently: V600G was the predominant mutation in primary tumors (5.3%), while V600E was found in metastases (15.4%).

The combined use of ddPCR and NGS improves the accuracy of molecular characterization of mCRC and is essential for personalized therapy selection.

PREVALENCE, INFECTION INTENSITY, AND ALLERGENIC RISK OF ANISAKIS SPP. IN READY-TO-EAT FISH PRODUCTS

2026-05-04T23:04:25+05:00

Anisakis spp. are foodborne parasites of major public health concern due to their dual role as infectious agents and potent allergens. This study investigated the prevalence, infection intensity, and allergenic potential of Anisakidae in ready-to-eat fish products available on the Astana market (Kazakhstan). A total of 27 commercial fish product brands representing various species and processing methods were examined using artificial digestion and molecular analysis. Third-stage larvae (L3) of Anisakis were detected in 10 products, including cod liver, pink salmon, tuna, mackerel, smoked herring, lightly salted herring, and smoked mackerel. Infection prevalence ranged from 7.5% to 100%, while mean infection intensity varied markedly, with the highest values observed in smoked and lightly salted herring products. PCR assays targeting the 18S rRNA and COX II genes confirmed the presence of Anisakidae DNA in all parasitologically positive samples. Notably, several products without detectable larvae by artificial digestion tested positive by PCR, indicating the persistence of Anisakis-derived genetic material associated with thermostable allergenic proteins. These findings demonstrate that the absence of visible larvae does not necessarily indicate the absence of allergenic risk and highlight the importance of combining parasitological and molecular methods for comprehensive food safety assessment of ready-to-eat fish products.

DEVELOPMENT AND OPTIMIZATION OF AN ELISA TEST SYSTEM FOR THE QUANTITATIVE DETERMINATION OF ALPHA-FETOPROTEIN

2026-03-27T14:33:34+05:00

This study presents the development of a prototype enzyme-linked immunosorbent assay (ELISA) test system for the quantitative determination of human alpha-fetoprotein (AFP) using monoclonal antibodies (mAbs). Hybridoma cell lines 1E3 and 1C7 were recovered after long-term storage, followed by cultivation, recloning, and evaluation of antibody production stability. Both 1E3 and 1C7 lines were shown to maintain stable secretion of highly specific anti-AFP monoclonal antibodies.

Preparative quantities of antibodies were obtained both in vitro and in vivo. The concentration of mAbs exceeded 1 mg/mL, and their specific activity was confirmed by indirect ELISA. Based on mAbs 1C7 and 1E3, key parameters of a sandwich ELISA were optimized, including selection of the capture antibody, determination of the optimal immobilization concentration, and establishment of the working dilution of the horseradish peroxidase-conjugated detection antibody. mAb 1C7 was selected as the capture antibody, as it provided higher signal intensity and improved linearity of the calibration curve.

The developed test system demonstrated a specific correlation between optical density and AFP concentration within the studied range, confirming the functional suitability of the selected antibody pair for the development of a diagnostic kit. The obtained results provide a foundation for further optimization of assay conditions aimed at improving analytical sensitivity and reproducibility.

ADDRESSING A MANUFACTURING BOTTLENECK IN CAR-T THERAPY: SCALABLE PRODUCTION OF TRANSFECTION-GRADE PLASMID DNA FOR CAR-T VECTOR MANUFACTURING

2026-08-24T10:34:52+05:00

The rapid advancing of cell and gene therapy has increased the demand for plasmid DNA used in viral vector manufacturing. The production of transfection-grade plasmid DNA remains a critical bottleneck, particularly for academic and emerging manufacturing facilities, due to the high cost and limited scalability of existing purification technologies. The aim of this study was to develop a practical and cost-effective approach for producing transfection-grade plasmid DNA suitable for lentiviral vector manufacturing.

The described acidic phenol extraction method was evaluated using plasmids commonly employed in lentiviral packaging systems and compared with a reference method, CsCl density gradient ultracentrifugation. The resulting plasmid preparations showed efficient removal of genomic DNA and other impurities, including endotoxin – the acidic phenol method consistently reduced endotoxin levels to 1.4–3.8 EU/µg DNA. Moreover, the purified plasmid DNA was predominantly in the supercoiled (covalently closed circular) form. Functional validation in transfection assays confirmed that plasmids purified by the proposed method supported lentiviral vector production with efficiency comparable to the reference method, yielding titers exceeding 5×10⁶ TU/mL, with no statistically significant differences in transduction efficiency between the groups.

These findings confirm that the acidic phenol extraction method is a simple, scalable, and economical approach for isolating transfection-grade plasmid DNA for lentiviral vector production. The method represents a practical alternative to ultracentrifugation-based purification and may facilitate broader use of cell and gene therapy by reducing dependence on specialized equipment and costly commercial kits.

PRODUCTION OF RACTOPAMINE-SPECIFIC ANTIBODIES FOR THE DETECTION OF GROWTH PROMOTER RESIDUES IN MEAT PRODUCTS

2026-08-21T15:12:48+05:00

Ractopamine is a non-steroidal β-adrenergic growth promoter widely used as a feed additive in intensive livestock production in developed countries to improve feed conversion efficiency, reduce adipose tissue deposition, and enhance lean muscle growth. However, residual ractopamine accumulated in animal tissues may pose significant risks to human health. Consequently, the use of ractopamine is prohibited in member states of the Eurasian Economic Union and the European Union. Nevertheless, residues of this compound may be present in livestock products imported from United States, Canada, Mexico, and Brazil.

The aim of this study was to generate highly specific antibodies against ractopamine for the development of an immunoassay-based method for detecting ractopamine residues in meat products. Successful synthesis of ractopamine conjugates with high-molecular-weight protein carriers enabled the production of polyclonal and monoclonal antibodies exhibiting strong specificity toward ractopamine in indirect ELISA. The analytical sensitivity achieved in competitive ELISA using both buffer solutions and meat matrixes demonstrated that the obtained monoclonal and polyclonal antibodies can serve as highly specific immunoreagents for the effective monitoring of ractopamine residues in meat products.

DEVELOPMENT AND OPTIMIZATION OF A TARGETED NGS PANEL FOR MODY DIAGNOSIS

2026-07-30T10:00:05+05:00

This study presents the development and optimization of a targeted next-generation sequencing (NGS) panel for the diagnosis of maturity-onset diabetes of the young (MODY). MODY is frequently misdiagnosed as type 1 or type 2 diabetes, highlighting the need for accurate genetic testing approaches.

This study aimed to design a primer panel targeting MODY-associated genes, optimize multiplex PCR conditions, and evaluate the performance of the developed workflow.

A total of 124 primer pairs were designed using PCRpanel software and tested. Specific amplification of target regions (300-400 bp) was confirmed by agarose gel electrophoresis. Multiplex PCR conditions were optimized. DNA libraries were prepared and successfully sequenced using the Illumina MiSeq platform. The minimum sequencing coverage achieved was 30×, with most regions exceeding 100×.

The developed panel demonstrated high specificity and may serve as an effective diagnostic tool for MODY.

CHARACTERIZATION OF THE YELLOW FEVER VIRUS REPLICATION COMPLEX USING AN NS1–GFP REPLICON

2026-07-27T10:22:05+05:00

Yellow fever virus is a mosquito-borne flavivirus responsible for severe human disease. Flaviviruses cause an estimated 400 million infections annually worldwide and are transmitted between animals and humans via arthropod vectors. Viral RNA replication is mediated by non-structural proteins that assemble into membrane-associated replication complexes (RC). Among these, the NS1 protein plays a critical role in replication complex formation and function. In this study, we investigated the replication complex of Yellow fever virus using sucrose density gradient ultracentrifugation. An NS1–GFP replicon system was constructed and employed to isolate and fractionate RC-associated components. This approach enables characterization of the molecular composition and organization of the replication complex and provides insights into the mechanisms underlying RNA replication in flaviviruses.

INVESTIGATION OF SUBSTRATE SPECIFICITY AND KINETIC PARAMETERS OF ESTERASE FROM THE SPOROGENIC STRAIN BACILLUS PARALICHENIFORMIS T7

2026-05-21T09:01:23+05:00

Esterases represent an important class of hydrolase enzymes widely utilized in biotechnology due to their high catalytic activity, substrate specificity, and stability against various physico-chemical factors. The sporogenic strain Bacillus paralicheniformis T7 produces extracellular enzymes, including esterase, which are associated with sporulation processes and adaptation to stressful conditions. In this study, the substrate specificity and catalytic properties of a recombinant esterase from the sporogenic strain B. paralicheniformis T7, expressed in Escherichia coli cells, were investigated. Structural analysis revealed that the esterase belongs to the GDSL/SGNH-hydrolase family and contains a conserved Ser–Asp–His catalytic triad. Substrate specificity analysis using p-nitrophenyl esters (C2–C18) showed that the enzyme exhibits maximum activity toward short-chain substrates, specifically p-nitrophenyl acetate (250.0 ± 0.1 U/mg), classifying it as a true esterase. The study of various factors demonstrated that divalent metal ions Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺, Fe²⁺ inhibit enzyme activity, while the reducing agents DTT and β-mercaptoethanol completely suppress its catalytic function. At the same time, the enzyme shows relative stability toward non-ionic detergents and sodium dodecyl sulfate (SDS). Kinetic analysis revealed that the K_m value for p-nitrophenyl acetate is 26.18 mM, V_max is 52.37 U/mg, and the catalytic constant k_cat is 1.74 s-1. The results indicate a distinct affinity of the enzyme for the substrate and moderate catalytic efficiency compared to other bacillar esterases. Thus, the esterase from B. paralicheniformis T7 is characterized by a pronounced specificity for short-chain esters and resistance to a range of surfactants, determining its potential for application in the biocatalytic synthesis of esters used in the food and fragrance industries.

MOLECULAR GENETIC CHARACTERIZATION AND OPTIMIZATION OF EX VITRO ADAPTATION AND ACCLIMATIZATION OF ENDANGERED BERBERIS ILIENSIS M. POP. MICROSHOOTS

2026-06-09T12:33:21+05:00

The conservation of rare and endangered plant species requires the development of effective approaches for obtaining viable planting material suitable for ex situ preservation and subsequent reintroduction. Berberis iliensis M. Pop. is a rare endemic species with limited natural regeneration, which necessitates the application of biotechnological methods. The aim of this study was to obtain viable planting material of Berberis iliensis using in vitro culture followed by adaptation and ex vitro acclimatization. Microshoots were established from shoot explants and cultured on Murashige and Skoog (MS) medium supplemented with growth regulators. Rooting was induced on MS medium containing indole-3-butyric acid (IBA). The effect of substrate composition on plant adaptation was evaluated. The highest growth and development parameters were obtained on a soil:sand substrate, where shoot height reached 6.53±0.14 cm, the number of leaves was 6.30±0.23, root number was 4.93±0.14, and root length was 5.11±0.20 cm, with all differences being statistically significant compared to the control. During acclimatization, the influence of mineral nutrition was assessed. The most pronounced positive effect was observed with calcium nitrate treatment, resulting in maximum plant height (16.84±0.42 cm), number of leaves (20.45±0.61), and crown diameter (15.83±0.62 cm). The results demonstrate that the successful transition of in vitro-derived plants to ex vitro conditions depends on substrate properties and mineral nutrition, which affect plant growth and survival. The proposed approach enables the production of viable planting material and can be applied for conservation and restoration of Berberis iliensis populations.

STATISTICAL, MOLECULAR, AND ULTRASTRUCTURAL INSIGHTS INTO THE STRESS-ALLEVIATING POTENTIAL OF AUTOCHTHONOUS STEPPE BACTERIAL LINEAGES

2026-06-22T17:57:12+05:00

The identification and characterization of multi-stress tolerant Plant Growth-Promoting Bacteria (PGPB) are essential for developing sustainable agricultural strategies to mitigate the effects of abiotic stress factors and intensive agrochemical usage. This study aimed to evaluate the stress tolerance of eleven autochthonous strains isolated from steppe zone previously exposed to glyphosate application. Taxonomic identity was confirmed by 16S rRNA gene sequencing, resulting in a phylogenetically diverse collection of isolates belonging to the genera Pseudomonas spp., Paenibacillus spp., and Paenarthrobacter spp. The strains were subjected to a comparative tolerance screening under abiotic and xenobiotic stress, including osmotic, saline, and glyphosate exposure. Quantitative growth histograms revealed distribution of strains depending on stress factors, while principal component analysis (PCA) identified distinct clustering patterns, indicating genus-specific tolerance thresholds. Molecular screening detected the groL and ectA genes in Paenibacillus isolates, indicating potential roles in protein folding and osmoadaptation. Furthermore, ultrastructural scanning and transmission microscopic imaging revealed exopolysaccharide (EPS) production, suggesting its role in cellular protection. Overall, the eleven indigenous strains showed synergistic physiological, genetic, and structural adaptations, highlighting their potential as multi-functional bio-inoculants for stress-resilient agriculture.

RAPID DETECTION OF THE BLATEM GENE IN ACINETOBACTER BAUMANNII USING RPA ISOTHERMAL AMPLIFICATION AND THE CRISPR/CAS12A SYSTEM

2026-09-11T14:34:57+05:00

Antibiotic resistance is one of the key problems of modern medicine, especially in relation to nosocomial infections caused by Acinetobacter baumannii, which has a high ability to accumulate drug resistance genes. One of the beta-lactam antibiotic resistance genes is blaTEM, which plays an important role in the formation of resistance in gram-negative microorganisms, including Acinetobacter baumannii. Standard molecular genetic methods for detecting antibiotic resistance genes, including PCR and real-time PCR, require the use of thermal cycling equipment. In this regard, the development of isothermal amplification methods with alternative methods of recording the result is of interest to laboratories with limited instrumentation. In this regard, the use of isothermal amplification methods in combination with CRISPR/Cas systems for sensitive detection of target genes is a promising direction. The purpose of this study was to develop and evaluate the sensitivity of a method for detecting the blaTEM gene based on recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system. The study showed that the RPA method provides specific amplification of the target gene with a detection limit of 10⁴ copies/reaction. The results obtained confirm the fundamental operability of the RPA-CRISPR/Cas12a system for detecting a fragment of the blaTEM gene and fluorescence registration of the amplification product.

PRODUCTION OF RECOMBINANT LACTASE FROM Lactobacillus helveticus VA FOR APPLICATION IN ADVANCED MILK PROCESSING TECHNOLOGIES

2026-08-24T07:59:23+05:00

β-Galactosidases (lactases, EC 3.2.1.23) are widely applied in the production of lactose-free dairy products and whey processing. The aim of the study is to obtain recombinant β-galactosidase and to determine its biochemical and kinetic parameters.

In the present work, eight strains were isolated from goat, sheep, mare, and camel milk, as well as cheeses and fermented dairy products from the Akmola and Almaty regions: Lactobacillus plantarum NAR, Lactobacillus plantarum VGM1, Lactobacillus paracasei VK2, Lactococcus lactis ZCM, Lactococcus lactis IS2, Lactobacillus curvatus ICH3, Lactobacillus rhamnosus ICH5, and Lactobacillus helveticus VA.

Screening for lactase activity revealed that the cheese-isolated strain Lactobacillus helveticus VA exhibited the highest activity compared to the other strains, reaching 212.63 U/mg. The β-galactosidase gene from Lactobacillus helveticus was cloned into the pET-28c(+) vector, and the recombinant lactase rLacLM was produced via plasmid expression in the Escherichia coli BL21(DE3) strain. Using immobilized metal affinity chromatography (IMAC), rLacLM was purified from the induced recombinant culture. The recombinant β-galactosidase exhibited maximum activity at pH 6.5–7.0 and a temperature of 50°C. The specific activity of the enzyme was 76.1 U/mg, while the Km, max, kcat, and kcat/Km values were 21.76 mM, 1015 µM·min⁻¹, 141.48 s⁻¹, and 6.5 × 10³ M⁻¹·s⁻¹, respectively. Thin-layer chromatography confirmed efficient lactose hydrolysis yielding glucose and galactose.

These results indicate that the recombinant β-galactosidase from Lactobacillus helveticus VA possesses biochemical and catalytic characteristics compatible with milk processing conditions, rendering it a promising enzyme for the production of lactose-free dairy products and the further development of domestic enzyme preparations.

CRYOPRESERVATION OF SAIGA (SAIGA TATARICA) SOMATIC CELLS: EFFECT OF NON-EQUILIBRIUM AND EQUILIBRIUM FREEZING ON FIBROBLAST VIABILITY

2026-09-24T16:46:20+05:00

Background. The saiga antelope (Saiga tatarica) has recovered numerically in Kazakhstan, but its populations remain exposed to mass mortality events that can remove a large share of a population within weeks. Viable somatic cell banking can insure genetic diversity, yet no cryopreservation protocol had been reported for saiga cells. Aim. To establish primary fibroblast cultures from saiga ear skin and to compare post-thaw survival after passive and controlled-rate slow freezing. Methods. Ear skin biopsies were collected from 50 adult saigas of the Betpak-Dala and Ustyurt populations. Fibroblasts obtained by explant culture were frozen at passage 2 either passively in cryovials with 10% dimethyl sulfoxide (DMSO; ≈ −1 °C/min to −70 °C) or in 0.5 mL straws with 1.5 M DMSO or 1.5 M ethylene glycol (EG) after 2 h equilibration at 5 °C, using a programmable freezer. Viability was assessed by trypan blue exclusion. Results. Cell outgrowth began on days 6–10 of culture. Passive freezing reduced viability from 97.6% to 44.5% (p < 0.001), with high inter-individual variability (9–77%; CV 30%). Controlled-rate freezing yielded 52.8 ± 1.1% viable cells with DMSO and 62.1 ± 1.0% with EG versus 85.8 ± 0.8% in fresh controls (p < 0.001); EG exceeded DMSO by 9.3 percentage points and gave 27% more viable cells per straw. Both controlled-rate treatments outperformed passive freezing and showed 2.9–3.5-fold lower variance. Conclusions. Controlled-rate slow freezing with 1.5 M EG was the most effective protocol and was adopted for the first somatic cell cryobank of the saiga in Kazakhstan.

VITRIFICATION OF SAIGA (SAIGA TATARICA) SKIN FIBROBLASTS: EFFECT OF CRYOPROTECTANT COMPOSITION AND ULTRA-RAPID COOLING AT −205 °C

2026-09-23T17:06:37+05:00

Background. The saiga antelope (Saiga tatarica) remains exposed to recurrent mass mortality, and a bank of viable somatic cells can insure its genetic diversity. Vitrification of saiga cells has not been studied. Aim. To establish fibroblast lines from neonatal saiga calves and to compare three vitrification solutions under two cooling regimens. Methods. Ear skin explants from 50 calves (25 males, 25 females) aged up to two weeks from the Betpak-Dala region were cultured in DMEM. Cells were loaded stepwise with 20% dimethyl sulfoxide (DMSO), 20% ethylene glycol (EG) or 20% DMSO + 20% EG, each with 0.5 M sucrose, and cooled in 0.25 mL straws either in liquid nitrogen (−196 °C) or in subcooled nitrogen at −205 °C (super-cooling ultra-rapid vitrification, SCURV). Viability was assessed by trypan blue exclusion (n = 50 lines per group). Results. Cell outgrowth began on days 6–10 of culture. Two-way ANOVA showed effects of both the cooling regimen (p < 0.001; η² = 0.19) and the solution (p < 0.001; η² = 0.04), without interaction. SCURV increased viability by 6.1 percentage points (95% CI 4.7–7.5), and the DMSO + EG mixture by 3.1 points relative to either single agent (p = 0.001); DMSO and EG did not differ. The highest viability, 53.9 ± 5.9%, was obtained with DMSO + EG under SCURV. Conclusions. The DMSO + EG mixture combined with ultra-rapid cooling at −205 °C was the most effective vitrification protocol. Samples of all 50 calves were added to the saiga somatic cell cryobank.

EFFECT OF CONDITIONED MEDIUM FROM 3D SPHEROIDS OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS ON ENDOTHELIAL CELL MIGRATION IN AN IN VITRO WOUND-HEALING MODEL

2026-09-23T15:37:02+05:00

Three-dimensional (3D) spheroid culture may modulate the paracrine activity of mesenchymal stem cells. This study compared the effects of conditioned media from two-dimensional (2D) cultures and 3D spheroids of human umbilical cord mesenchymal stem cells (hUC-MSCs) on endothelial wound closure in vitro. Passage-3 hUC-MSCs were cultured as adherent monolayers or assembled into spheroids using MicroTissues® agarose micromolds. Spheroid morphology and diameter were assessed at 24, 48, and 72 h. Immunofluorescence analysis included CD73, CD90, CD105, and CD45 in 2D cultures and CD90 and CD105 in intact 3D spheroids. Human umbilical vein endothelial cells (HUVECs) were exposed to 2D-conditioned medium (2D-CM), 3D-conditioned medium (3D-CM), or endothelial growth medium (control). Scratch-assay images were acquired at 0 and 24 h, and wound closure was calculated relative to the initial cell-free area. Spheroids formed in all 81 assessed microwells. Mean diameter increased from 310 ± 20 µm at 24 h to 420 ± 25 µm at 48 h and 500 ± 30 µm at 72 h (mean ± SD). CD90 and CD105 remained detectable following spheroid formation. At 24 h, wound closure estimated from individually selected images (not from independent experiments) was approximately 100%, 66.0%, and 72.7% for 3D-CM, 2D-CM, and control, respectively. These preliminary descriptive findings suggest greater endothelial wound closure with 3D-CM; confirmation requires matched-field analysis across independent experiments with statistical analysis.

A PRACTICAL WORKFLOW FOR MASCOT-BASED PROTEIN IDENTIFICATION AND POST-TRANSLATIONAL MODIFICATION ANALYSIS

2026-07-14T11:30:09+05:00

Modern biological research has become highly interdisciplinary, requiring mass spectrometry data generation to be combined with bioinformatics and data analysis skills to obtain meaningful results. However, many researchers who regularly conduct mass spectrometry experiments often lack practical experience in interpreting results and optimizing database searches. This practical article describes the use of the Mascot search engine for identifying peptides and proteins in shotgun proteomics. Using a local Mascot server (version 2.6.2), two datasets were analyzed: an in-house Escherichia coli tryptic lysate obtained on an Impact II QTOF mass spectrometer and a publicly available histone post-translational modifications dataset from the jPOST repository. The workflow includes optimizing search parameters, performing the search, interpreting results, and demonstrating the detection of post-translational modifications. The presented example of histones demonstrates the identification of known lysine modifications (including trimethylation) detected through search. This article serves as a practical guide for researchers wishing to improve their proteomic data analysis skills in Mascot and highlights the critical importance of selecting the right parameters to obtain accurate and reproducible results.

DEVELOPMENT OF A PCR TEST SYSTEM FOR THE GENETIC DIAGNOSIS OF HEMORRHAGIC FEVER WITH RENAL SYNDROME

2026-09-10T15:00:25+05:00

Hemorrhagic fever with renal syndrome (HFRS) is an acute zoonotic disease caused by orthohantaviruses. Reliable molecular diagnostics are essential for early detection and epizootiological surveillance. This study aimed to develop and validate a reverse transcription quantitative real-time PCR (RT-qPCR) assay for detection of HFRS-associated orthohantavirus RNA.Species-specific primers (HV P1-F and HV P1-R) and a TaqMan probe (HV P1) targeting the nucleocapsid protein (N) gene within the S segment were designed. A plasmid positive control containing a 242-bp target fragment was constructed. Analytical sensitivity, specificity, reproducibility, and repeatability were evaluated. Field testing included samples from 343 small mammals collected in endemic areas of the West Kazakhstan Region. The assay consistently detected the plasmid control from 1 ng/µL to 1 fg/µL, with Ct values of 37.35 and 37.73 at 1 fg/µL. No specific amplification was observed at 100 ag/µL. A strong linear relationship between Ct values and template concentration was obtained (R² = 0.998). No false-positive reactions occurred with tick-borne encephalitis virus or Crimean-Congo hemorrhagic fever virus RNA. Reproducibility and repeatability showed 100% concordance of qualitative results. HFRS-associated orthohantavirus RNA was detected in 45 of 123 pooled field samples. The minimum infection prevalence was 13.1%, reaching 15.3% in Myodes glareolus and 13.3% in Apodemus uralensis and Microtus arvalis. The developed one-step RT-qPCR assay demonstrated high sensitivity, specificity, and reproducibility and is suitable for routine diagnostics and surveillance of natural HFRS foci.

IODINE COORDINATION COMPOUNDS OF ANTI-INFLAMMATORY DRUGS: NEW SYSTEMS OF INTEREST FOR ANTI-INFECTIVE RESEARCH

2026-08-10T16:24:31+05:00

Given the increasing threat of antimicrobial resistance and multidrug-resistant pathogens, the development of combined therapeutic systems with preserved anti-infective activity and supportive anti-inflammatory properties is high-ly relevant. This study aimed to synthesize and evaluate novel iodine-containing coordination compounds based on flunixin meglumine and dexamethasone sodium phos-phate. The synthesized compounds were characterized using capillary electrophoresis and UV-spectroscopy. Their cytotoxicity was assessed in PBMC culture, while antimicrobial and antiviral activities were evaluated against bacterial, fungal, and viral pathogens. Two stable compounds, KC-247 and KC-249, were suc-cessfully obtained. Both demonstrated antimicrobial activity against Staphylococcaceae, Pseudomonadaceae, Enterobacteriaceae, Enterococcaceae, and yeast-like fungi. MBC values ranged from 0.801–0.200 µg/mL for KC-247 and 2.093–0.523 µg/mL for KC-249. In PBMC culture, CC₅₀ values were 0.072 mg/mL and 0.225 mg/mL, respectively. KC-249 showed pronounced antiviral activity against influenza A (H1N1), while KC-247 demonstrated moderate effects. Neither compound was active against herpes simplex virus type 1. KC-247 and KC-249 exhibited promising an-timicrobial and selective antiviral properties with acceptable cytotoxicity, highlighting their potential as multifunctional candidates for novel anti-infective therapeutics.

COMPARISON OF THE EFFECTIVENESS OF THE EXPERIMENTAL KIT DEVELOPED FOR EXOSOME ISOLATION COMPARED WITH A COMMERCIAL KIT IN RELEVANT MODELS

2026-09-21T12:40:28+05:00

Background: Exosomes derived from mesenchymal stem cells (MSCs) are of considerable interest for regenerative medicine; however, their application is limited by the lack of standardized and reproducible isolation methods.

Objective: To compare the efficiency of an experimental kit and a commercial kit for exosome isolation from the conditioned medium of human umbilical cord-derived MSCs.

Methods: Exosomes were isolated from conditioned medium collected from MSCs at passages 4 and 10 and from different medium volumes (1, 5, and 9 mL). Particle size and zeta potential were determined by particle tracking analysis, while total protein concentration was measured using the Bradford assay.

Results: Both kits enabled the isolation of vesicles of comparable size characteristic of exosomes (161.4–177.9 nm). The commercial kit provided a higher protein yield, whereas the experimental kit differed in the magnitude and polarity of the zeta potential. For both methods, the yield decreased at later MSC passages and increased with larger volumes of conditioned medium.

Conclusion: The experimental kit provides isolation of exosome-like extracellular vesicles comparable to that obtained with the commercial kit and may be considered a promising alternative, provided that additional assessment of preparation purity and molecular markers is performed.

OBTAINING OF AN EXPERIMENTAL RETINOPATHY MODEL IN RABBITS USING REPEATED INTRAVITREAL ADMINISTRATION OF VEGF AND BFGF

2026-09-23T15:39:33+05:00

Retinopathy is one of the causes of early vision loss among the working population. Retinopathy is a common complication of diabetes. However, preclinical testing of new therapies in animal models of diabetic retinopathy remains complicated by the long time to symptom onset and the high cost of animal housing. One of the solutions is to create models similar to retinopathy without inducing diabetes. One of the approaches for such method is to induce retinopathy using factors such as vascular endothelial growth factor and basic fibroblast growth factor. In our study, we tested the ability of a combination of these factors at low concentrations to induce a stable retinopathy model. The results showed that, despite the fact that the concentration of the factors was reduced, repeated administration of them resulted in a model exhibiting all the key features of the disease.

DEVELOPMENT AND PRELIMINARY ANALYTICAL EVALUATION OF REAL-TIME PCR ASSAYS FOR AFRICAN SWINE FEVER VIRUS DETECTION

2026-06-17T11:55:23+05:00

African swine fever virus (ASFV) is a large, double-stranded DNA virus that poses a major threat to the pig industry, highlighting the need for reliable molecular diagnostics. This study reports on the design and analytical development of two primer–probe systems targeting conserved ASFV genomic regions (B646L/p72), supported by in silico assessment and laboratory testing with synthetic DNA constructs. Classical PCR confirmed amplification of both targets from cloned control templates, producing amplicons of the expected size. In probe-based real-time PCR, consistent amplification was obtained for the ASFV-3 system using a pET-asf-3 clone across serial dilutions (Ct ~27–34), while the ASFV-1 probe-based assay did not generate a detectable signal under the applied conditions. Under the tested conditions, the preliminary limit of detection observed for the ASFV-3 probe-based assay was 10³ copies per reaction. These results demonstrate the feasibility of the developed approach and indicate that the ASFV-3 system supports further analytical evaluation, while the ASFV-1 system requires additional optimization of probe parameters and/or thermal cycling conditions.

EVALUATION OF THE BIOLOGICAL ACTIVITY OF ORTHOPOXVIRUS STRAINS IN PRIMARY CHICKEN EMBRYO FIBROBLASTS

2026-06-12T16:25:56+05:00

Understanding the biological features of the genus, Orthopoxvirus, including their replicative activity, is essential for their application in virology and vaccinology. Comparative analysis of biological activity in cell cultures makes it possible to evaluate the replicative potential of strains and the degree of their adaptation to the cellular substrate.

The aim of the study was to standardize a method for obtaining primary chicken embryo fibroblasts (CEF) and to use them as an in vitro substrate for assessing the biological activity of various Orthopoxvirus strains, with an emphasis on cytopathic effect (CPE) and infectious activity. Vaccinia virus, cowpox virus, and camelpox virus were used in the study. Primary CEF were obtained from 9-11-day embryonated chicken eggs (ECE), the optimal seeding concentration for the formation of a monolayer substrate was determined, and the cultures were infected with the viruses under study; CPE and virus titer were assessed during sequential passages. The results showed that a seeding concentration of 5×10⁵ cells/mL ensured the formation of a uniform CEF monolayer within 24 h. Upon infection, all three viruses induced similar CPE, characterized by cell rounding, loss of elongated morphology, and partial destruction of the monolayer, with the main difference being the timing of CPE onset. Vaccinia virus strains induced CPE within 24 h, whereas cowpox virus and camelpox virus induced CPE after 36 and 48 h, respectively. Both vaccinia virus strains exhibited high infectious activity and rapid adaptation (titers from 5.50 to 6.58±0.12 lg TCID₅₀/mL), whereas cowpox and camelpox viruses showed a lower and stable titer (titers from 5.50 to 6.58±0.12 lg TCID₅₀/mL), and delayed CPE development.

The results emphasize the significance of CPE onset time and infectious titer level as key parameters for evaluating replicative capacity and strain adaptation, which is important for laboratory studies and the selection of vaccine candidates.

COMPARATIVE STUDY OF ELISA STOP SOLUTIONS BASED ON MINERAL ACIDS

2026-08-12T12:12:06+05:00

A comparative study of ELISA stop solutions prepared on the basis of hydrochloric, orthophosphoric and sulfuric acids was performed using a competitive ELISA model for detection of antibodies against bluetongue virus. It was demonstrated that the chemical nature and concentration of the acid significantly affect the optical density of negative (K-) and positive (K+) controls, the time-dependent change of the signal, and the ratio between negative and positive controls.

Hydrochloric acid solutions within the range of 0.5-4.0 M provided a high initial signal for K- (approximately 2.0 OD units), limited optical density variation over 30 minutes (3-8%), and the highest control ratio values (50-90). Orthophosphoric acid resulted in an increase of the positive control signal over time, whereas sulfuric acid caused a decrease in both K- and K+ signals, leading to narrowing of the dynamic range.

The findings confirm that the selection of the stop solution is a critical factor in ELISA standardization and directly influences the analytical reliability of the diagnostic test system.

PRODUCTION OF HYPERIMMUNE SERUM AGAINST RECOMBINANT PROTEINS SODC AND OMP16 OF BRUCELLA SPP. FOR THE DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TEST SYSTEM

2026-09-10T14:21:00+05:00

Brucellosis is a widespread zoonotic disease transmitted from domestic animals to humans and is endemic in many regions of the world. There is a need for highly specific serological tests for brucellosis based on recombinant proteins to reduce cross-reactivity with other Gram-negative bacteria and to differentiate between vaccinated and infected individuals. The aim of the present study was to obtain hyperimmune sera against Brucella spp. antigens in laboratory animals. Rabbits and guinea pigs were used as experimental animals. For immunization, recombinant Brucella spp. proteins Omp16 and SodC were used in combination with adjuvants, including aluminum hydroxide (Al(OH)₃), as well as complete and incomplete Freund’s adjuvant (CFA/IFA). Immunization was performed according to a standard protocol, followed by monitoring of antibody levels in blood sera using enzyme-linked immunosorbent assay (ELISA) and agar gel precipitation test (AGPT). For protein expression, producer strains were cultured in LB medium supplemented with kanamycin. Protein synthesis was induced with 0.1 mM IPTG. Purification was performed using immobilized metal affinity chromatography (IMAC).

As a result of the study, hyperimmune sera with a high level of specific antibodies were obtained. It was demonstrated that the use of adjuvants promotes the development of a pronounced humoral immune response in immunized animals. The highest antibody titers were observed in animals immunized with CFA/IFA adjuvants, where antibody titers reached 1:6400 in rabbits and 1:1600 in guinea pigs. When aluminum hydroxide was used, antibody titers were 1:3200 and 1:800 in rabbits and guinea pigs, respectively.

The obtained results confirm the effectiveness of the proposed approach for producing hyperimmune sera and demonstrate their potential for use as reference positive controls in the development and standardization of serological methods for brucellosis diagnostics.

LABORATORY-SCALE CULTIVATION OF THE OSPA-VAKTSINA VACCINIA VIRUS STRAIN IN VERO CELLS ON BIONOC II® MACROCARRIERS USING THE BELLOSTAGE™-3000 SYSTEM

2026-09-02T12:32:24+05:00

Vaccinia virus remains relevant as a viral substrate for smallpox vaccines and as an orthopoxvirus model. This study evaluated, at laboratory scale, the feasibility of cultivating the “Ospa-vaktsina” vaccinia virus strain in Vero cells grown on BioNOC II® macrocarriers using the BelloStage™-3000 Tide Motion system. The study was not designed as a comparative assessment of BelloStage™-3000 performance against another cultivation platform.

Vero cells were cultivated in BelloCell®-500A vessels in Dulbecco’s Modified Eagle Medium supplemented with 2% fetal bovine serum. After 120 h of cell cultivation, when the total cell number reached 1.99 × 10⁹ cells per vessel, cultures were infected with a working virus inoculum at a calculated infectious input of 0.01 TCID₅₀ per cell. The infectious input was calculated as the total TCID₅₀ delivered in the 500-mL inoculum divided by the total cell number at infection. One bioreactor run included three parallel BelloCell®-500A vessels (n = 3 culture units within a single run).

Cell attachment to the macrocarriers was 89% at 3 h after seeding. The total cell number increased from 7.45 × 10⁷ cells at seeding to 1.99 × 10⁹ cells per vessel at 120 h, corresponding to a 26.7-fold increase. Following infection, the virus titer increased from 4.60 log₁₀ TCID₅₀/mL in the working inoculum to a maximum of 6.00 log₁₀ TCID₅₀/mL at 120 h post-infection, a 1.40-log₁₀ (approximately 25.1-fold) increase. Culture medium parameters were maintained within predefined operating ranges for the cell-growth and post-infection phases.

Under the conditions tested, the Vero/BioNOC II® culture supported replication of the “Ospa-vaktsina” strain in BelloStage™-3000. Because the study comprised a single bioreactor run and did not include a parallel comparator platform, the findings demonstrate laboratory-scale feasibility but do not establish technological superiority, process scalability, or suitability for vaccine manufacturing.

OPTIMIZATION OF SERUM-FREE CULTIVATION OF RECOMBINANT SHEEPPOX VIRUS SSPV (TK) OMP16 IN VERO CELLS

2026-06-23T13:25:50+05:00

Brucellosis remains a major zoonotic disease of global public health and veterinary importance. Recombinant capripoxvirus vectors have emerged as promising platforms for the development of next-generation vaccines owing to their favorable safety profile, genomic stability, and ability to induce robust immune responses. The aim of this study was to optimize serum-free cultivation conditions for the recombinant sheeppox virus strain SSPV (TK) OMP16 expressing the Brucella outer membrane protein OMP16 in Vero cell culture.

The recombinant virus was propagated on Vero cell monolayers using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum or serum-free OptiPro™ medium containing 1% L-glutamine. Adaptation to serum-free conditions was achieved by five sequential blind passages in Vero cells. Viral biomass accumulation was evaluated at multiplicities of infection (MOI) of 0.01, 0.05, 0.1, and 0.5 TCID₅₀/cell during cultivation periods ranging from 12 to 120 h. Infectious activity was determined by TCID₅₀assay in Vero cell culture.

Serum-free OptiPro medium supported efficient replication of the recombinant virus and produced infectious titers comparable to those obtained in conventional serum-containing DMEM. The highest infectious activity of recombinant strain SSPV (TK) OMP16 was 6.25 log TCID₅₀/cm³ at an MOI of 0.1 TCID₅₀/cell at 96 h post-infection. Sterility testing confirmed the absence of bacterial and fungal contamination in all virus suspensions.

The optimized serum-free cultivation protocol provides a standardized approach for propagation of recombinant capripoxvirus vectors and represents a practical basis for further development and scale-up of recombinant vaccine production against brucellosis.

VALIDATION OF KASP MARKERS FOR ADAPTATION AND YIELD-RELATED TRAITS IN SPRING BREAD WHEAT BREEDING LINES OF KAZAKHSTAN

2026-07-14T08:58:07+05:00

The validation of molecular markers under target environmental conditions is essential for the effective implementation of marker-assisted selection in wheat breeding. This study aimed to assess the genetic diversity of spring bread wheat germplasm from Kazakhstan, validate KASP markers associated with adaptation and yield-related traits, and identify favorable alleles for breeding applications. A total of 107 spring bread wheat genotypes originating from five breeding organizations were evaluated in four contrasting environments of Kazakhstan and genotyped using 43 KASP markers developed from previous genome-wide association studies (GWAS) and quantitative trait locus (QTL) mapping studies. Of the analyzed markers, 33 were polymorphic and successfully differentiated the studied germplasm. Genetic diversity analysis revealed moderate variation among breeding collections, with the highest level of polymorphism observed in the lines from Karabalyk Agricultural Experimental Station collection (100%). Principal coordinate analysis demonstrated clear genetic differentiation among breeding programs. Association analysis identified numerous significant marker–trait relationships across environments, with the largest number detected in North Kazakhstan. Five markers (ipbb_ta_152, ipbb_ta_219, ipbb_ta_223, ipbb_ta_183, and ipbb_ta_162) showed stable associations with adaptation- and yield-related traits across all testing locations. Evaluation of allelic effects revealed that marker ipbb_ta_162 had the strongest positive influence on grain yield, particularly under North Kazakhstan conditions, whereas ipbb_ta_183 exhibited the most stable favorable allele across environments. The results confirm the usefulness of KASP markers for marker-assisted selection and provide valuable molecular tools for the development of high-yielding and environmentally adapted wheat cultivars in Kazakhstan.

A DETECTION OF FIELD NEWCASTLE DISEASE VIRUS GENOTYPE VII AND INFECTIOUS BURSAL DISEASE VIRUS IN A COMMERCIAL LAYING FLOCK IN THE AKMOLA REGION

2026-09-15T16:23:44+05:00

Infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) remain important pathogens in commercial poultry production. In vaccinated flocks, molecular detection requires careful interpretation because viral genetic material may originate from either field infection or vaccine-associated signals. We investigated a mortality episode in a vaccinated commercial laying flock in the Akmola Region using reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing of selected viral genomic fragments. Five deceased laying hens were examined. NDV RNA was detected in all five birds by targeting the L gene, and sequencing supported its assignment to genotype VII and a field-associated rather than vaccine-lineage sequence. IBDV RNA was detected in two of the five birds by targeting VP2; sequencing identified a field-associated sequence most closely related to GenBank accession D00869.2.2 within the analyzed fragment. The findings demonstrate concurrent detection of genetically distinct field-associated NDV and IBDV sequences within the investigated mortality episode. Given the very small sample size and the absence of quantitative virological, serological, and detailed pathological data, the study does not establish the causal contribution of either virus to the observed production losses or mortality, nor does it demonstrate flock-level co-circulation. The results nevertheless illustrate the value of sequence-based verification when interpreting molecular detections in vaccinated poultry.

DEVELOPMENT AND STANDARDIZATION STRATEGY FOR QUALITY CONTROL OF A RECOMBINANT CAPRIPOXVIRUS-VECTORED VACCINE AGAINST BRUCELLOSIS

2026-09-09T12:50:39+05:00

This work presents the development and implementation of a quality control and standardization strategy for a recombinant capripoxvirus-vectored vaccine against brucellosis. We conducted a comprehensive analysis of World Health Organization guidelines, the State Pharmacopoeia of the Republic of Kazakhstan, and regulatory documents governing quality control of biological medicinal products. Based on this analysis, we established a comprehensive panel of quality parameters encompassing physicochemical, molecular-genetic, biological, and microbiological characteristics of the vaccine, as well as assessment of residual process-related impurities. Using the developed strategy, we evaluated three pilot batches of the recombinant capripoxvirus-vectored brucellosis vaccine.

The results demonstrated that all pilot batches met the established quality and safety requirements. The developed quality control and standardization strategy provides a foundation for further refinement of the evaluation system for recombinant vector-based brucellosis vaccines and for the preparation of regulatory documentation to support their clinical application.

OPTIMIZATION OF GENOME-WIDE SNP MICROARRAY DATA PROCESSING FOR GENERATION OF AN ATHLETE GENETIC PROFILE

2026-10-06T17:05:51+05:00

Based on the analysis of global experience and our own preliminary studies aimed at identifying associations between athletic performance potential and individual genotypes across 97 SNP polymorphisms (75 candidate genes), interpretations of possible genotype variants for an athlete’s genetic passport were developed. A software program and several auxiliary scripts were created using the RStudio development environment and the Python programming language. The developed tools efficiently create and update databases containing the required genetic information, reducing the time needed to develop a genetic passport while minimizing the risk of errors. These tools significantly improved the efficiency of storing, sorting, and processing primary data obtained from genome-wide SNP microarray genotyping. The reduction of human involvement in the repetitive process of collecting and comparing tables minimized the risk of errors and facilitated the analysis of genotyping data. Automation of primary and secondary processing of genome-wide SNP microarray genotyping data, genotype interpretation, database updating, and generation of completed genetic passport reduced data processing time from several months to 20-30 minutes.

PREPARATION OF EFFECTIVE DIAGNOSTIC REAGENTS SUITABLE FOR ELISA IN THE DIAGNOSIS OF BACILLUS ANTHRACIS

2026-08-05T18:32:18+05:00

The aim of this study was to optimize methods for precipitating gamma-globulins from hyperimmune sera obtained from animals immunized with Bacillus anthracis strains, followed by the preparation of immunoperoxidase conjugates for use in enzyme-linked immunosorbent assays (ELISA).

Two methods for gamma-globulin isolation were compared: ammonium sulfate precipitation and multi-step ethanol fractionation according to a modified Kon method. Protein yield, purity, and precipitating activity in the radial diffusion in gel (RDG) assay were evaluated. The results showed that the ethanol method provided higher gamma-globulin yield (up to 85 %) and protein concentration (17.4 mg/mL) compared to ammonium sulfate precipitation. Maximum precipitating activity (1:16) was achieved using the optimized ethanol precipitation, whereas the activity obtained with ammonium sulfate was 1:8.

The isolated fractions were conjugated with horseradish peroxidase using the periodate oxidation method. Spectrophotometric analysis at 280 nm and 403 nm demonstrated that conjugates prepared from ethanol-precipitated gamma-globulins exhibited more uniform enzymatic activity and stable optical properties.

These results indicate that ethanol fractionation is an effective approach for obtaining immunoperoxidase conjugates suitable for ELISA-based detection of Bacillus anthracis.

A REVIEW OF THE CLINICAL TRANSLATION OF NANOEMULSIONS, DOXIL AND LIPID NANOPARTICLES

2026-08-24T07:57:18+05:00

Nanomedicine has reshaped the clinical use of therapeutics by changing how payloads are formulated, protected, distributed and released. Yet despite decades of innovation, only a limited number of nanocarrier platforms have achieved sustained clinical impact. Here, we examine nanoemulsions, Doxil and lipid nanoparticles (LNPs) as three clinically validated platforms spanning increasing levels of carrier dependence. Nanoemulsions primarily solve formulation and administration constraints. Doxil reprograms the pharmacokinetics and toxicity of an established drug. LNPs enable nucleic acid therapeutics whose clinical activity depends on carrier-mediated protection, delivery and intracellular release. This reveals a central principle of translational nanomedicine: clinical value emerges when carrier function addresses an indispensable therapeutic need, rather than when nanoscale complexity is pursued as an end in itself. We further discuss how biological identity, tissue transport, cellular processing, payload release and manufacturability determine whether this functional advantage is retained in patients. Framing nanomedicines according to therapeutic dependence on the carrier provides a clinically grounded basis for understanding past successes and guiding the design of the next generation of translatable nanotherapeutics.

STABILITY OF AN ELISA TEST SYSTEM BASED ON A RECOMBINANT CHIMERIC SM PROTEIN FOR THE DETECTION OF ANTIBODIES TO SARS-COV-2 UNDER DIFFERENT TEMPERATURE CONDITIONS

2026-09-11T15:17:50+05:00

Assessment of the stability of kit components is a critical step in the development of enzyme-linked immunosorbent assay (ELISA) systems, as it directly determines the accuracy and reproducibility of diagnostic results during storage and routine use. In the present study, the stability and reproducibility of a newly developed ELISA test system based on the recombinant chimeric SM protein were evaluated under different temperature conditions. The stability of the kit components was assessed by monitoring changes in the analytical signal, expressed as optical density (OD), and the variability of replicate measurements, expressed as the coefficient of variation (CV, %), over a controlled period at -20ºC, 4ºC, 22ºC, and 37ºC. The results demonstrated that the stability of the developed ELISA test system was strongly temperature-dependent. Standard refrigerated storage at 4ºC was identified as the optimal condition, providing the highest preservation of the analytical signal, with 81.3% of the initial signal retained (OD₄₅₀ = 1.933), while maintaining high reproducibility (CV = 1.74%), consistently below the predefined acceptance threshold of ≤5%. Storage at -20ºC showed notable limitations. Despite maintaining a relatively high analytical signal (OD₄₅₀ = 1.623) and 68.2% residual activity, freezing and thawing of the reagents resulted in increased analytical variability (CV = 5.56%), exceeding the established acceptance criterion. Exposure to elevated temperatures of 22ºC and 37ºC had a pronounced detrimental effect on test-system stability, resulting in degradation of protein components and a loss of more than 65% of the initial activity. These findings demonstrate that, to ensure long-term preservation of the sensitivity, specificity, and reproducibility of the developed ELISA diagnostic kit, storage at 4ºC under properly maintained cold-chain conditions is an essential technological requirement.