Competitive Enzyme Immunoassay Research Articles

Ochratoxin a levels in Turkish coffee: a probabilistic health risk assessment via Monte Carlo simulation.

Throughout history, Turkish coffee has been the most widely consumed type of coffee in Turkey. Therefore, the goal of this study was to determine the amount of ochratoxin A (OTA) present in Turkish coffee and to analyze any potential health hazards. A total of 41 Turkish coffees were collected and analyzed for OTA activity using a competitive enzyme immunoassay (ELISA). Furthermore, dietary exposure and health risk assessments for the Turkish population were calculated based on analytical results and coffee consumption data from the Turkish Nutrition and Health Survey 2019 (TNHS-2019) in three age categories (15-18, 19-64, and 65 + years). Nine of the samples contained more than 2.5μg/kg OTA, with an average of 5.24μg/kg. The OTA concentration in 3 samples exceeded the permissible maximum limit (5μg/kg) established by Turkish legislation, and the mean concentration was 8.41μg/kg. A margin of exposure (MOE) approach was used for risk characterization, considering both non-neoplastic and neoplastic consequences. There were no concerns about health risks because MOEs were more than 10,000 for all categories. Although the levels of OTA analyzed in Turkish coffee did not pose a risk to individuals in the three age categories, emphasis should be placed on minimizing and controlling OTA concentrations in Turkish coffee. Additionally, it is also necessary to consider other food sources that could contribute to OTA exposure.

Ramadan fasting and exercise combination therapy: A novel approach for osteoporosis prevention in ovariectomized rats.

Osteoporosis is a chronic bone metabolic disease that affects millions of people worldwide, particularly the elderly and postmenopausal women. It is characterized by weakened bones, increasing the risk of fractures and leading to significant morbidity and mortality. The goal of the current study is to examine the reported osteo-preservative effects of exercise and/or fasting in the Ramadan fasting model (RFM) in ovariectomized (OVX) rats. The experimental intervention started 1month following the ovariectomy procedure and consisted of five 15-min exercise sessions per week at 18-25m/min and/or an approximately 13-h fast from sunrise to sunset (6:00 AM-19:00 PM). Serum bone metabolism biomarker levels were measured, and mineral concentrations in femoral ashed bones and digested serum were determined. Additionally, serum bone alkaline phosphatase (b-ALP), parathyroid hormone, osteocalcin, calcitonin, and vitamin D3 concentrations were measured using the competitive enzyme immunoassay technique. Calcium, magnesium, and phosphorus showed a notable decrease in mineral concentration among OVX rat femurs compared with the combination group (OVX + RFM + E) and control groups. In addition, homeostasis of serum concentrations of calcium, magnesium, and phosphorus was observed to increase in the OVX + RFM + E group rather than in the OVX group without intervention when compared with a control group. Furthermore, fasting and exercise, either alone or concurrently with ovariectomy, induced a non-significant elevation in osteocalcin, parathyroid hormone, and vitamin D3, whereas b-ALP and calcitonin increased significantly compared with those in control rats. The combination of the Ramadan fasting model and moderate intensity exercises among OVX rats manifested advantageous effects in bone biomarkers compared with OVX rats without intervention. This could be recommended as a lifestyle modification that is protective against osteoporosis, especially in the context of depleted estrogen hormone after menopause.

Биологически активные гидролизаты белков молока и их комплексы включения с циклодектринами

Enzymatic hydrolysis of dairy proteins increases their nutritional and biological value while reducing their allergenic potential. The subsequent complexation of peptides with cyclodextrins (CDs) reduces the bitterness of the hydrolyzed proteins. The research objective was to obtain hydrolysates of whey proteins and their cyclodextrin inclusion complexes with peptides, as well as to describe the peptide composition of the cleaved dairy proteins, biological activity, and sensory profile of the hydrolysates and inclusion complexes. The research featured enzymatic whey protein hydrolysates with an extensive hydrolysis degree and their inclusion complexes with β- and γ-CDs. Dairy proteins were hydrolyzed with alcalase, and the hydrolysates obtained were subjected to micro- and ultrafiltration (cut-off limit – 10 kDa). The peptide composition of the hydrolyzed proteins was determined by the methods of high-performance liquid chromatography and chromatography-mass spectrometry. The antimutagenic activity was evaluated using the Ames test whereas the antibacterial effect was studied with the impedimetric method. The antioxidant activity was detected with fluorimetry and spectrophotometry. The method of competitive enzyme immunoassay was applied to reveal the antigenic properties. The bitterness of the experimental sample s was determined by a sensory evaluation. The research delivered the optimal modes for whey protein cleavage with alcalase that made it possible to achieve efficient micro- and ultrafiltration. The resulting hypoallergenic peptide fractions and their inclusion complexes with β- and γ-CDs possessed antioxidant, antibacterial, and antimutagenic properties. The whey proteolysis and subsequent filtration with/without tindalization demonstrated a 265/589-fold decrease in the residual antigenicity. The fluorimetric method showed a 1.79/1.90-fold increase in the antioxidant activity of the hydrolysate in complexes with β- and γ-CDs. Binding of β-CDs to peptides enhanced their antimicrobial activity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538. The hydrolysate samples with β-CDs showed less bitterness. Whey proteolysis with alcalase under optimized conditions and subsequent fractionation resulted in a product with high consumer qualities. Enzymatic hydrolysates of dairy proteins and their CD inclusion complexes were able to substitute native protein components. Their bioactive properties, good taste, and low allergenic potential mean good prospects for the functional food industry.

Mycotoxins in vegetating corn plants from experimental mono-sowing

The aim of present study was to reveal the mycotoxin contamination of vegetative maize (Zea mays L.) plants during the periods of leaf formation and panicle emergence. Early maturing hybrids of the varieties Krasnodarsky 194 MV, Ladozhsky 175 MV and Competence®, resistant to fungal diseases, were grown in the spring-summer period of 2023 on the experimental field of the Russian State Agrarian University – Moscow Agricultural Academy (Moscow) with sod-podzolic soil and the application of NPK fertilizers 16:16:16. For mycotoxicological analysis, aerial parts of plants were collected weekly from the phase of formation of the 3rd leaf (18 days after sowing. Samples of seedlings, leaves and stems (total number – 172), after drying, were ground in a laboratory mill and extracted with a mixture of acetonitrile and water in a volumetric ratio of 84:16 with a consumption of 10 ml per 1 g of sample. Mycotoxin content was determined in extracts after 10-fold dilution with phosphate-salt buffer solution pH 7.5 by indirect competitive enzyme immunoassay. T-2 toxin, deoxynivalenol, diacetoxiscirpenol, roridin A, sterigmatocystin and PR toxin were absent in the samples. Corn seedlings and leaves contained cyclopiazonic acid (CPA), emodin (EMO), mycophenolic acid (MPA), alternariol (AOL), ergot alkaloids (EA), aflatoxin B1 (AB1) and single samples – zearalenone (ZEN), ochratoxin A (OA) and citrinin (CIT) in concentrations from 16 up to 35 μg/kg. Fumonisins of group B are found only in early seedlings. The permanent contaminant of the stems was MPA, whereas EA and CIT were absent. In the seedlings and leaves of all hybrids, the detection of AOL and EA remained stable during the change of development phases, as did the average concentrations of CPA (about 100 μg/kg), AOL (from 17 to 27 μg/kg), EMO (35–58 μg/kg), MPA (28–41 μg/kg), EA (6–18 μg/kg) and AB1 (2 μg/kg). In the stems of plants in phases 7–9 of leaf and sweeping, variation in cases of detection of CPA, EMO, AOL, AB1 and OA was noted by varieties.

The use of 1E12, a monoclonal anti-platelet factor 4 antibody, to improve the diagnosis of vaccine-induced immune thrombotic thrombocytopenia

BackgroundVaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-CoV-2 due to prothrombotic immunoglobulin (Ig) G antibodies to platelet factor 4 (PF4) and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin. ObjectivesWe assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context. MethodsThe ability of F(ab′)2 fragments of 1E12, 1C12, and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine-scanning mutagenesis was performed to define the amino acids involved in the interactions between the monoclonal antibodies and PF4. ResultsA strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition, 93%; n = 8), whereas it had no effect on the binding of HIT antibodies (median, 6%; n = 8). In contrast, 1C12 and 2E1 inhibited VITT (median, 74% and 76%, respectively) and HIT antibodies (median, 68% and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition. ConclusionA simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.

Plasmonic enzyme immunoassay via nanobody-driven controllable aggregation of gold nanoparticles for detection of ochratoxin A in pepper

Ochratoxin A (OTA) in food poses a serious challenge to public health. Herein, using the nanobody-driven controllable aggregation of gold nanoparticles (AuNPs) in a glucose oxidase-tyramine-horseradish peroxidase (GOx-TYR-HRP) system, we propose a direct competitive plasmonic enzyme immunoassay (dc-PEIA) for OTA detection. The OTA-GOx conjugate catalyzes glucose to produce hydrogen peroxide (H2O2), and then HRP catalyzes H2O2 to generate hydroxyl radical which induces the crosslink of TYR. Crosslinked TYR leads to aggregation of AuNPs through strong electrostatic interactions, which is tunable based on the competition of OTA-GOx and free OTA for binding the immobilized nanobody. The optimized dc-PEIA achieves an instrumental limit of detection (LOD) of 0.275 ng/mL and a visual LOD of 1.56 ng/mL. It exhibits good selectivity for OTA and accuracy in the analysis of pepper samples, with the confirmation of high-performance liquid chromatography. Overall, the dc-PEIA is demonstrated as a useful tool for detecting OTA in food.

Prolidase could be considered a sign of inflammation associated with cigarette smoking.

Smoking causes inflammation, thickening, and narrowing of the airways. This inflammatory process is a reaction to free radicals and oxidants. Smoking affects collagen metabolism and tissue remodeling. Prolidase enzyme hydrolyzes iminodipeptides with hydroxyproline and C terminal proline. It plays a crucial role in the metabolism of collagen and the remodeling of the matrix. The present study aims to reveal the association of prolidase with inflammation caused by smoking and to compare serum prolidase levels with oxidative-antioxidative status in healthy individuals. A total of 76 participants (38 smokers and 38 nonsmokers) were involved in the present study. Serum cotinine levels were measured to show the exposure to nicotine in tobacco smoke by using the competitive inhibition enzyme immunoassay method. Serum prolidase, total oxidant status (TOS), and total antioxidant status (TAS) were determined by the enzyme-linked immunosorbent (ELISA) method, respectively. The correlation between smoking, serum prolidase levels, TOS, and TAS was investigated. TAS and serum prolidase levels of smokers were considerably lower than those in non-smokers (p < 0.001, p = 0.012 respectively). However, no differences were observed in TOS between the two groups. There was no statistically significant correlation between serum prolidase levels, TAS, and TOS. Moreover, no relationship was observed between respiratory function parameters and serum prolidase levels. To the best of our knowledge, the present study is the first study to demonstrate the role of prolidase in smoking-related inflammation. The results achieved in the present study suggest that smoking creates an imbalance in the oxidant-antioxidant activity. Smoking decreases prolidase levels, leading to decreased collagen turnover. Chronic pulmonary disease might be related to this decrease in collagen turnover.

Both coiling and clipping induce the time-dependent release of endogenous neuropeptide Y into serum.

The vaso- and psychoactive endogenous Neuropeptide Y (NPY) has repeatedly been shown to be excessively released after subarachnoid hemorrhage and in numerous psychiatric disorders. NPY is stored in sympathetic perivascular nerve fibers around the major cerebral arteries. This prospective study was designed to analyze the impact of microsurgical and endovascular manipulation of the cerebral vasculature versus cranio- and durotomy alone on the serum levels of NPY. 58 patients (drop-out n = 3; m:f = 26:29; mean age 52.0 ± 14.1 years) were prospectively enrolled. The vascular group underwent repair for unruptured intracranial aneurysms (UIA) of the anterior circulation [endovascular aneurysm occlusion (EV) n = 13; microsurgical clipping (MS) n = 17]; in the non-vascular group, 14 patients received microsurgical resection of a small-sized convexity meningioma (CM), and 11 patients with surgically treated degenerative lumbar spine disease (LD) served as control. Plasma was drawn (1) before treatment (t0), (2) periprocedurally (t1), (3) 6 h postprocedurally (t2), (4) 72 h postprocedurally (t3), and (5) at the 6-week follow-up (FU; t4) to determine the NPY levels via competitive enzyme immunoassay in duplicate serum samples. We statistically evaluated differences between groups by calculating one-way ANOVA and for changes along the time points using repeated measure ANOVA. Except for time point t0, the serum concentrations of NPY ranged significantly higher in the vascular than in the non-vascular group (p < 0.001), with a slight decrease in both vascular subgroups 6 h postprocedurally, followed by a gradual increase above baseline levels until FU. At t3, the EV subgroup showed significantly higher NPY levels (mean ± standard deviation) than the MS subgroup (0.569 ± 0.198 ng/mL vs. 0.415 ± 0.192 ng/mL, p = 0.0217). The highest NPY concentrations were measured in the EV subgroup at t1, t3, and t4, reaching a climax at FU (0.551 ± 0.304 ng/mL). Our study reveals a first insight into the short-term dynamics of the serum levels of endogenous NPY in neurosurgical and endovascular procedures, respectively: Direct manipulation within but also next to the major cerebral arteries induces an excessive release of NPY into the serum. Our findings raise the interesting question of the potential capacity of NPY in modulating the psycho-behavioral outcome of neurovascular patients.

Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein

A sensitive chemiluminescent enzyme immunoassay (CLEIA) was established for the determination of gentamicin (GEN) residue levels in animal tissue. This assay is based on a fusion protein of single-chain variable fragment (scFv) and alkaline phosphatase (AP). Initially, VL and VH derived from anti-gentamicin monoclonal antibody were linked by a short peptide to construct a scFv. Subsequently, the constructed scFv sequence was accessed into the pLIP6/GN vector, and a soluble scFv-AP fusion protein was generated. The scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) for the determination of gentamicin. In the dcCLEIA, the half inhibitory concentration (IC50) and limit of detection (LOD) were 1.073 ng/mL and 0.380 ng/mL, respectively. The average recoveries of gentamicin spiked in animal tissue samples ranged from 78 % to 96 %. These results showed a strong correlation with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The above results suggest that the anti-GEN scFv-AP fusion protein is suitable for detecting gentamicin residues in edible animal tissues.

Influence of mother’s genotype on the concentration and variability of triiodothyro- nine in landrace pigs

One of the leading roles in the hormonal system of the body of pigs in terms of functional significance is played by the thyroid gland, which, through the hormone triiodothyronine, has a complex effect on all systems, organs and physiological processes while controlling the metabolism and energy in the animal’s body. The impact of maternal genotype on the concentration and variability of triiodothyronine in complete siblings of Landrace pigs was studied. For the study, blood serum samples were taken from 33 pigs using the vacuum method from the jugular vein. Blood samples were taken according to generally accepted practices. The studies were carried out in the laboratory of the biochemistry of the Novosibirsk State Agrarian University using the technique of competitive enzyme immunoassay with a set of reagents “Steroid ELISA-triiodothyronine” to quantify the concentration of triiodothyronine in blood serum. Enzyme immunoassay was performed on a Thermo Scientific Multiskan FC analyser. In pig breeding, the content of heavy metals in water, soil, feed, organs and tissues was constantly monitored. The concentration of chemical elements did not exceed the MPC level. Statistical processing of experimental data was carried out using standard methods of descriptive statistics and the statistical programming language or data analysis environment RStudio (using the functions: descrstats, summary, sd, read.table, write. table, aes). The strength of the influence of the genotype of mothers of the Landrace breed on the triiodothyronine content in complete siblings in the conditions of Western Siberia was established, which amounted to 4.6 %. In some mothers’ offspring, the triiodothyronine level was 1.9 and 1.8 times higher than in others. Two clusters of mothers were identified according to the concentration of triiodothyronine in complete siblings, which included three groups of mothers with an average level of 2.26 nmol/l and five groups with the attention of 1.30 nmol/l. The reference limits of the triiodothyronine level in Landrace pigs in the conditions of Western Siberia were established.

Development of an immunoassay based on a specific antibody for the detection of diphenyl ether herbicide fomesafen

Fomesafen belongs to the diphenyl ether herbicide, and is widely used in the control of broadleaf weeds in crop fields due to its high efficiency and good selectivity. The residual of fomesafen in soil has a toxic effect on subsequent sensitive crops and the microbial community structure because of its long residual period. Therefore, an efficient method for detecting fomesafen is critical to guide the correct and reasonable use of this herbicide. Rapid and sensitive immunoassay methods for fomesafen is unavailable due to the lack of specific antibody. In this study, a specific antibody for fomesafen was generated based on rational design of haptens and a sensitive immunoassay method was established. The half maximal inhibitory concentration (IC50) of the immunoassay was 39 ng/mL with a linear range (IC10–90) of 1.92–779.8 ng/mL. In addition, the developed assay had a good correlation with the standard UPLC-MS/MS both in the spike-recovery studies and in the detection of real soil samples. Overall, the developed indirect competitive enzyme immunoassay reported here is important for detecting and quantifying fomesafen contamination in soil and other environmental samples with good sensitivity and high reproducibility.

Fecal metabolite and blood steroid concentration patterns in relation to reproductive behavior during the ovarian cycle of African lions (Panthera leo)

To develop successful ex-situ breeding programs for the conservation of threatened wildlife, an understanding of reproductive endocrinology in the species of interest is essential. Fecal hormone metabolite patterns are widely used in this regard, but limited information is available regarding how these reflect blood hormone profiles. This study aimed to longitudinally monitor and compare circulating progestagen (sP) and estrogen (sE) concentrations, as well as fecal progestagen (fPM) and estrogen (fEM) metabolite concentrations with observed reproductive behaviors of African lionesses. For 18 months, blood sampling (n = 309) from five captive lionesses took place 1–7 times per week during positive reinforcement training. In parallel, over a period of 9 months, fecal samples (n = 129) from four of these lionesses were searched for daily and collected when found. Daily behavioral monitoring of all females enabled classification of reproductive stage according to presence/absence of characteristic behavioral events (i.e., “allowing mount”, “copulation”, “flirting run”, “lordosis”, “purring”, and “rolling”). Competitive enzyme immunoassays were used for steroid quantification. In total, 11 an-ovulatory and 16 ovulatory cycles (7 pregnancies and 9 pseudo-pregnancies) were assessed. When compared, the pattern of fecal hormone metabolite concentrations matched reproductive behavior more reliably than circulating steroid concentrations. Both sP and fPM patterns were correlated (n = 51; r = 0.86) and enabled identification of luteal phases, which helped to distinguish between pregnant and non-pregnant females. In contrast, detection of estrus by measurement of sE and fEM was not accurate regardless of the matrix evaluated.

Quantitative determination of prothioconazole in wheat grain, soybean, and pond water based on a polyclonal antibody.

Prothioconazole and its metabolite are considered a potential threat to human health and environmental safety. Thus, the development of a sensitive and rapid detection method for prothioconazole is crucial to ensure the safety of agricultural products. In this study, a new hapten of prothioconazole was designed and synthesized, and a selective polyclonal antibody with high affinity against prothioconazole was produced, which was obtained from immunized New Zealand white rabbits. Based on the polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) were developed for detecting prothioconazole pesticides. Under optimized experimental conditions, the limit of quantification (LOQ) values for ic-CLEIA and ic-ELISA were 1.8 and 10.7 ng mL-1, respectively. The results demonstrated that the sensitivity (LOQ) achieved by ic-CLEIA was more than five times higher compared to that obtained with ic-ELISA. In addition, the recoveries obtained by adding standard prothioconazole to wheat grain, soybean, and pond water samples were in the range of 81.9 to 104.7% for ic-ELISA and 89.0 to 118.0% for ic-CLEIA.

The Effect of the Supplementation of Humic Substances and Fermented Products in the Feed on the Content of Salinomycin Residues in Poultry Tissues.

The presence of antimicrobial residues in products of animal origin is a constant problem for consumer health. The aim of this study was to observe the effect of the addition of humic substances (H), fermented products (F) and a mixture of both (FH) to feed supplemented with the coccidiostat salinomycin, compared with a control group (C), on the content of salinomycin residues in the edible tissues of broiler chickens using two microbial inhibition screening methods, Explorer 2.0 test and the Screening Test for Antibiotic Residues (STAR), and a confirmatory competitive enzyme immunoassay analysis (Salinomycin ELISA Kit). The results of the microbial inhibition tests showed a gradual decline in the positive results in the tissue samples from the last day of salinomycin administration (30th day) tothe last day of fattening (37th day, day of slaughter) in group C and no positive results in the tissue samples from experimental groups H, F and FH slaughtered on the last day of fattening. Using the Salinomycin ELISA Kit, salinomycin was detected in the chicken muscle tissues of all the control and experimental groups. However, no sample from any group contained salinomycin at a concentration exceeding the maximum residue limits set by European law. The high level of significance (p < 0.001) confirmed the positive influence of the administration of humic substances and fermented products on the content of salinomycin residues in chicken tissues.

Prevalence of hepatitis D virus infection among patients with chronic hepatitis B infection in a tertiary care centre in Thailand

Knowledge about the epidemiology of hepatitis D virus (HDV) is essential for effective screening and management. Our study aimed to update the prevalence of HDV infection among patients with hepatitis B virus (HBV) infection at hepatology clinics in Thailand. We enrolled HBV-infected patients from hepatology clinics at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between June 2022 and November 2023. Demographic, biochemical characteristics, and liver-related complications (LRC), including cirrhosis and hepatocellular carcinoma, were reviewed. The competitive enzyme and chemiluminescence immunoassays were used to detect anti-HDV antibodies. Real-time polymerase chain reaction (RT-PCR) was used to test for HDV RNA in anti-HDV-positive patients. The HDV genotype was identified in detectable HDV RNA samples. Of the 702 enrolled patients, four (0.6%) had positive and equivocal for both anti-HDV tests. Two (50.0%) of the four patients tested positive for HDV RNA and genotype 1 was identified; one had multiple risk factors. Anti-HDV seroprevalence was not significantly different between patients with and without LRC. In conclusion, HDV co-infection is less common in Thailand than globally. Additionally, our study identified genotype 1, the predominant HDV genotype worldwide, and observed co-infection even without LRC.

Blood oxytocin levels and the rs4686302 polymorphism of the oxytocin receptor gene in patients with adenomyosis

BACKGROUND: An in-depth study of the pathogenesis of adenomyosis and the development of new therapeutic approaches are extremely relevant. Atosiban, a competitive oxytocin receptor antagonist, may be a promising drug with a potential therapeutic effect. Oxytocin is involved in the pathogenesis of adenomyosis-associated dysmenorrhea, and there is evidence of increased expression of the oxytocin receptor (OXTR) in the myometrium in adenomyosis and the association of the OXTR gene polymorphic variant rs4686302 with oxytocin-induced myometrial contractility.&#x0D; AIM: The aim of this study was to evaluate blood oxytocin levels and the frequency of the polymorphic variants of the oxytocin receptor gene (OXTR) rs4686302 polymorphism in patients with adenomyosis and in healthy women of reproductive age.&#x0D; MATERIALS AND METHODS: The levels of oxytocin in the blood plasma were measured by competitive enzyme immunoassay (ELISA) in patients with adenomyosis (n = 56, with mean age of 37.5 ± 8.54 years), and in women without gynecological diseases (control group; n = 33, with mean age of 32.1 ± 5.40 years). The polymorphic variant rs4686302 of the OXTR gene was analyzed by PCR-RFLP analysis in patients with adenomyosis (n = 79, with mean age of 39.9 ± 7.74 years) and in control group patients (n = 49, with mean age of 37.0 ± 9.20 years). The chi-square (χ2) test was used to analyze the distribution of genotypes and alleles. Statistical data processing was performed using the Jamovi Software, Version 1.2.27 for Windows.&#x0D; RESULTS: In patients with adenomyosis, the level of oxytocin in the blood serum was significantly higher than in control group patients: 130.0 ± 27.9 pg/ml and 95.9 ± 26.7 pg/ml, respectively. The minor allele A of the OXTR gene polymorphic variant rs4686302 was significantly more common in patients with adenomyosis (28.5%) than in women in the control group (13.3%) and in the general sample (11–15% according to the 1000 Genomes Project). The odds of developing adenomyosis in patients with the presence of the A allele (the G/A and A/A genotypes) were 2.44 times higher compared to patients with the G/G genotype (OR 2.44, 95% CI 1.13–5.28).&#x0D; CONCLUSIONS: The data obtained suggest the participation of oxytocin and its receptor in the pathogenesis of adenomyosis and requires further research in this direction.

Preparation of monoclonal antibody and establishment of indirect competitive chemiluminescence enzyme immunoassay for sodium pentachlorophenolate

In this study, three haptens of sodium pentachlorophenolate (PCP-Na) were designed and synthesized, and the monoclonal antibody against PCP-Na with high specificity and affinity was successfully prepared. Based on this monoclonal antibody, a highly sensitive indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) was developed for the determination of PCP-Na and its metabolite pentachlorophenol (PCP). The ic-CLEIA method based on optimal conditions showed that the half maximal inhibitory concentration (IC50), linear range (IC20-IC80) and the detection limit (LOD, IC10) for PCP-Na were 0.083 ng/mL, 0.0076–1.5111 ng/mL, and 0.0032 ng/mL, respectively, and 0.29 ng/mL, 0.0983–0.7008 ng/mL, and 0.0192 ng/mL for PCP, respectively. The results of cross reaction showed that the method had no obvious cross reaction with other six chlorophenols compounds (phenol, 2-chlorophenol, 4-chlorophenol, 2, 4-dichlorophenol, 2, 4, 5-trichlorophenol, 2, 4, 6-trichlorophenol) except the metabolite PCP. The average recoveries in chicken, pork and fish samples were 81.3–105.8 %, with coefficient of variation (CV) lower than 7.6 %. In this study, a new highly rapid, sensitive and accurate ic-CLEIA method was established, which can be used for the sensitive detection of PCP-Na and its metabolite PCP in animal tissues.

Comparative analysis of IgG levels in blood sera from patients with COVID-19, persons vaccinated by «Gam-COVID-Vac» and healthy donors before the pandemic

Introduction. Serum IgG measurement is used in the diagnosis and monitoring of many diseases. Therefore, it seems important to study the impact of coronavirus pandemic on IgG levels in population and the role of this parameter in COVID-19.&#x0D; The aim of this study was to compare mean IgG levels in sera obtained from 31 hospitalized COVID-19 patients, 30 healthy donors before pandemic and 34 donors vaccinated with «Sputnik V» (have not had COVID-19).&#x0D; Materials and methods. Total IgG was quantitated by two enzyme immunoassays (EIAs): «IgG Total-ELISA-BEST» kit certificated in Russia and homemade competitive EIA utilizing bispecific monoclonal antibodies (bsAbs) against human IgG (HIgG) and horseradish peroxidase (HRP).&#x0D; Results and discussion. The groups did not show differences in IgG levels (regardless of sex) with both methods giving comparable results. However, «IgG Total-ELISA-BEST» kit revealed statistically significant differences in mean serum IgG levels in subgroups of male patients depending on the levels of antibodies to viral RBD-antigen: below and above 400 BAU/ml. In the first subgroup (10 men) the mean serum IgG content was 14.3 ± 4.1 mg/mL, while in the second (6 men) — 6.9 ± 2.7 mg/mL.&#x0D; Conclusion. Sera obtained before pandemic contained the same mean IgG concentrations as sera from donors vaccinated with «Sputnik V» and COVID-19 patients. The relatively decreased mean IgG concentration was found only in COVID-19 male patients with anti-RBD antibodies levels above 400 BAU/ml. In light of literature data on association of decreased serum IgG with COVID-19 severity, it would be reasonable to further compare larger groups, taking into account clinical differences. The possibility of using bsAbs for human Ig measurements by competitive EIA was demonstrated.

Pseudoneutralizing test for preclinical studies of vaccines against SARS-CoV-2

The purpose of this study was to evaluate efficiency of a competitive enzyme immunoassay which specifically detects antibodies that recognize the receptor-binding domain at the S1 subunit of SARS-CoV-2 coronavirus spike protein and block the formation of initiator infection complex between RBD and angiotensinconverting enzyme 2 (pseudo-neutralizing test, PNT) being applied at the stage of preclinical studies of anti- SARS-CoV-2 vaccine. We studied 37 animal blood sera (8 cows, 10 dogs) as well as 19 male and female transgenic mice of the B6.Cg-Tg(K18-ACE2)2Prlmn/HEMI line hemizygous for Tg(K18-ACE2)2Prlmn (Jackson Immunoresearch, West Grove, PA, USA)) immunized with candidate COVID-19 vaccine preparations containing SARS-CoV-2 Spike protein. In this study, 3 techniques were used for detection of antibodies to SARS-CoV-2 virus, as follows: 1) a pseudo-neutralizing test (PNT) to detect antibodies that block interaction between RBD and ACE-2; 2) neutralization test (RN) to detect virus-neutralizing antibodies, and 3) enzyme-linked immunosorbent assay to detect class G antibodies to RBD SARS-CoV-2. The results were expressed, respectively, as the suppression quotients (SC), titers of virus-neutralizing antibodies (VNA), and the positivity index (IP). The data obtained show a pronounced, statistically significant correlation between the results obtained by immunoassay methods with VNA titers determined in the studied animals by the virological neutralization test. E.g., the Spearman correlation quotients for VNA and SC titers, were, respectively, 0.9151; 0.8085, and 0.9207 for dogs, transgenic mice and cows. The Spearman quotient for VNA and PI titers was 0.8854 and 0.8955 for dogs and transgenic mice. Thus, in order to evaluate immunogenicity of vaccine preparations in our study, both methods are adequate and safe analogues to RN-ELISA for determination of IgG to RBD and PNT aoming for detection of antibodies blocking the formation of RBD/ACE-2 complex. However, the advantage of PNT is its versatility, eliminating the need to use different conjugates to detect antibodies in blood sera of different animal species. The data obtained for samples of three animal species (transgenic mice, dogs and cows) well agree with similar data obtained by us and other researchers for human blood sera, thus demonstrating high correlation between the results of PNT-like competitive tests to determine antibodies that block the formation of the RBD/ACE-2 complex, with VNA results in virologic neurtralization test (RN). Therefore, the proposed PNT technique may be used in preclinical and clinical trials of candidate vaccines and drugs.

Influence of Complexation with β- and γ-Cyclodextrin on Bioactivity of Whey and Colostrum Peptides.

Dairy protein hydrolysates possess a broad spectrum of bioactivity and hypoallergenic properties, as well as pronounced bitter taste. The bitterness is reduced by complexing the proteolysis products with cyclodextrins (CDs), and it is also important to study the bioactivity of the peptides in inclusion complexes. Hydrolysates of whey and colostrum proteins with extensive hydrolysis degree and their complexes with β/γ-CD were obtained in the present study, and comprehensive comparative analysis of the experimental samples was performed. The interaction of CD with peptides was confirmed via different methods. Bioactivity of the initial hydrolysates and their complexes were evaluated. Antioxidant activity (AOA) was determined by fluorescence reduction of fluorescein in the Fenton system. Antigenic properties were studied by competitive enzyme immunoassay. Antimutagenic effect was estimated in the Ames test. According to the experimental data, a 2.17/2.78-fold and 1.45/2.14-fold increase in the AOA was found in the β/γ-CD interaction with whey and colostrum hydrolysates, respectively. A 5.6/5.3-fold decrease in the antigenicity of whey peptides in complex with β/γ-CD was detected, while the antimutagenic effect in the host-guest systems was comparable to the initial hydrolysates. Thus, bioactive CD complexes with dairy peptides were obtained. Complexes are applicable as a component of specialized foods (sports, diet).