Enzyme Immunoassay Research Articles

The relationship between serum interleukin-1β and circulating CD16+ neutrophils for assessing progression-free time in advanced ovarian cancer

BACKGROUND: Ovarian cancer is the most aggressive gynecological tumor with high mortality. The ambivalent, both helper and suppressive effects of neutrophils on the cells of innate and adaptive immunity determines their important immunoregulatory role in the development of malignant tumors. AIM: Evaluation of the effect of serum interleukin-1β and circulating neutrophils CD16+ on progression-free time in advanced ovarian cancer. MATERIAL AND METHODS: In 42 primary patients with serous ovarian adenocarcinoma (median age 54 years) with ascites and 15 patients with benign ovarian tumors (median age 60 years) admitted for examination and treatment to the Regional Clinical Oncology Dispensary before receiving neoadjuvant antitumor treatment (2015–2020), the level of interleukin-1β in the blood serum was determined using enzyme immunoassay, and the number of CD16+ neutrophils was determined by fluorescence microscopy using monoclonal antibodies. Statistical processing was performed using Statistica 13 and Jamovi 2.4.14 programs. Progression-free time analysis of patients was performed using the Cox and Kaplan–Meier regression methods. RESULTS: It was found that the number of circulating CD16+ neutrophils in advanced ovarian cancer with ascites was lower than in benign ovarian tumors [48.0; 95% confidence interval (CI) 46.59–48.12 versus 50.0; 95% CI 48.51–49.76; p=0.017]. At the same time, the level of interleukin-1β in the blood serum in advanced ovarian cancer with ascites was higher than in benign ovarian tumors [2.52 (95% CI 2.50–3.37) versus 1.26 (95% CI 0.97–1.55) (p=0.0001)]. In multivariate Cox analysis in patients with ascites (odds ratio 4.334; 95% CI 1.83–10.23; p=0.001), both the CD16+ neutrophil count (odds ratio 0.73; 95% CI 0.62–0.86; p=0.0001) and serum interleukin-1β levels (odds ratio 1.90; 95% CI 1.41–2.57; p=0.0001) had prognostic significance for assessing progression-free time. CONCLUSION: The number of circulating CD16+ neutrophils and serum interleukin-1β levels simultaneously affect progression-free time in advanced ovarian cancer.

A-009 Analytical Characterization of a Novel NT-proBNP Assay on a Central Laboratory Platform

Abstract Background N-terminal pro-B-type Natriuretic Peptide (NT-proBNP) or B-type Natriuretic Peptide (BNP) assay(s) are widely measured with a Class 1, Level of Evidence A recommendation in professional heart failure (HF) guidelines for diagnosis and prognosis. We present the analytical characteristics of the first NT-proBNP assay on Beckman Coulter platforms. Methods Measurements were conducted with the Access NT-proBNP assay, a one-step sandwich immunoenzymatic assay using monoclonal capture and detection antibodies, on the DxI 9000 Immunoassay Analyzer with Lithium Heparin (LiHep) plasma samples. Performance was characterized according to CLSI guidelines for: Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ), linearity, imprecision (repeatability, reproducibility) and matrix comparison with LiHep, EDTA and serum. Reference intervals (RI) were evaluated in a healthy, adult U.S. population comprised of 675 subjects with 54.6% females, 79.1% White and 15.1% Black individuals. Exclusion criteria included elevated cardiac troponin, abnormal eGFR and cardiac medications. The 97.5th percentile RI upper limit was defined for age-stratified ranges of <50, 50-75, and >75 years old. Results Performance was LoB=1.1ng/L; LoD=4.8ng/L; LoQ=4.8ng/L; linearity=35,000ng/L. Repeatability ranged from 1.5%-3.5% and reproducibility from 2.3%-7.9% in seven samples ranging from 38ng/L to 23,848ng/L. Matrix measurement regression (n=68) yielded slopes of 1.00, 1.01; intercepts -0.03 to -2.63; r=0.98. Table 1 shows that NT-proBNP values increase with age and women generally have higher values than men. Conclusions The LoB, LoD and LoQ demonstrate highly sensitive low-end characteristics. Repeatability and reproducibility show excellent performance across a wide measurement range. NT-proBNP values are known to be age dependent, and the age-stratified 97.5th percentile RI upper limits were 162 ng/L for <50 years, 311 ng/L for 50-70 years, and 457 ng/L for >75 years. This novel Access NT-proBNP assay offers robust analytical performance as an aid to clinical HF management in acute settings.

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.

B-295 Sensitive and Rapid Homogeneous Immunoassay for the Detection of Zolpidem and its Major Metabolite in Urine

Abstract Background Zolpidem, a schedule IV controlled substance under the Controlled Substances Act, is commonly known as Ambien and Ambien CR, a prescription only treatment for insomnia. Zolpidem is one of the most common prescribed sleep aids in the U.S. due to its rapid sedative effects. The recommended duration of treatment is between two days and four weeks, as long-term use of zolpidem increases the risk of habit formation. Addiction to zolpidem can cause severe side effects, including memory loss, delusion, and in extreme cases, fatal overdose. From 2005-2010, Zolpidem-related emergency room visits doubled, underscoring the need for accurate testing as zolpidem testing plays an important role in cases of misuse, diversion, and forensics. The sole commercially available homogeneous immunoassay for the detection of zolpidem in urine has a relatively high cutoff at 20 ng/mL and poor cross-reactivity (0.02%) to major metabolite zolpidem 4-carboxylic acid. ARK Diagnostics has developed the ARK™ Zolpidem Assay to detect zolpidem at a cutoff concentration of 10 ng/mL with high cross-reactivity to its metabolite, Zolpidem phenyl 4-carboxylic acid and no cross-reactivity to zaleplon and zopiclone at concentrations below 100,000 ng/mL. Methods The ARK™ Zolpidem Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff concentration of 10 ng/mL and semi-quantitative range up to 40 ng/mL. Preliminary performance characterization for this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, analytical recovery, specificity, Histogram Overlap Analysis of ±50% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 2.6 to 3.1% CV. Spiked recovery of zolpidem ranged from 88.6% (2.5 ng/mL) to 96.8% (10 ng/mL). The major metabolite, zolpidem phenyl-4-carboxylic acid, showed an approximate equivalence to the 10 ng/mL zolpidem cutoff at 30 ng/mL (33.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 100% (15 positives and 100 negatives). Conclusions The ARK™ Zolpidem Assay measures zolpidem and its major metabolite zolpidem phenyl-4-carboxylic acid in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

B-055 Comparison of two different methods for quantitation of anti-dsDNA antibody

Abstract Background Anti-dsDNA antibody is a specific marker of rheumatic disorder, primarily systemic lupus erythematosus (SLE). Early detection of these diseases is most important in treatment and prognosis. We evaluated two different quantitation systems of anti-dsDNA antibody: the BioPlex 2200 multiplex simultaneous detect system and the single detection method, which is the Phadia 250 system. Methods 53 serum samples were analyzed by two different methods, which are BioPlex 2200, Bio-Rad Laboratories, Hercules, CA, and Padia 250, Thermo Fisher Scientific, Uppsala, Sweden, respectively. The method of the BioPlex 2200 system is multiplexed bead assay, and that of Phadia 250 is Fluorescence Enzyme Immunoassay (FEIA). Results The quantitation results of BioPlexx 2200 were 1 to 126 IU/mL, and those from Phadia 250 were 0.08 to 109.53 IU/mL, respectively. These two groups of the anti-dsDNA antibody values showed excellent correlations (r=0.9036). Conclusions The quantitation of anti-dsDNA antibody were performed mainly by a single analyzing method, Phadia 250, in Korea. Bioplex 2200 system is multiplex bead assay; it can analyze eight different Extractable Nuclear Antigen Antibodies (anti-ENA) simultaneously: Anti Ribosomal P Ab, Anti SSa/Ro Ab, Anti SSb/La Ab, Anti Centromere Ab, Anti Sm Ab, Anti RNP Ab, Anti SCL-70 Ab, Anti Jo-1 Ab as well. Nevertheless, these anti-ENA results showed semiquantitative results.

B-267 Validation of the Newly Developed Fully Automated Chemiluminescent Enzyme Immunoassay (CLEIA) Reagent for Measurement of Cyclosporine in the Blood

Abstract Background Cyclosporine (CsA) is an immunosuppressive drug requiring therapeutic drug monitoring (TDM) for its narrow-ranged medication level. Approximately 60% of CsA in the blood exist as molecular complexes with proteins like cyclophilin in lymphocytes and erythrocytes. Measurement of CsA in the blood by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires an extraction process for CsA from the whole blood specimen, which is often manual complicated and time-consuming process causing a risk of variation in the measurement values among different operators. In addition, the small (cyclic polypeptide composed of 11 amino acids) and hydrophobic property of CsA, make it difficult to establish antibodies for a sandwich immunoassay. In this study, we have validated a novel fully automated sandwich immunoassay reagent for measurement of blood CsA that used a combination of newly-established CsA antibodies and iTACT® (immunoassay for total antigen including complex via pretreatment) technology. Methods The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE® L2400 (FUJIREBIO INC). Twenty uL whole blood specimens are sampled after mixed with suction/discharge stirring to prevent that blood cells settle to the bottom of a container on the analyzer, and then treated with a pretreatment solution to obtain free CsA. The treated samples are assayed by a two-step sandwich immunoassay. Limit of Detection (LoD), Limit of Quantitation (LoQ), within-laboratory precision, linearity, no interference, and the correlation of this assay and LC-MS/MS were validated, following the current CLSI guidelines protocols. Data were analyzed using Microsoft Excel statistical tool Analyse-it. Results The LoD and the LoQ were 2.7 ng/mL and 10.8 ng/mL respectively. The within-laboratory precision showed CV 1.6-2.9 %. Linearity was demonstrated over the range 13.1-2381.5 ng/mL. No interference was observed with unconjugated (≤19.7 mg/dL) or conjugated bilirubin (≤20.1 mg/dL), chyle (≤1480 FTU), RF (rheumatoid factor, ≤1000 IU/mL), and HAMA (human anti-mouse antibody, ≤1010 ng/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.0 and 1.0, respectively (N=120). Conclusions The performance of our novel CsA assay, which use LUMIPULSE L2400, was satisfactory for routine analysis. A unique fully-automated process, including the pretreatment process of whole blood specimens, for measurement would contribute the low LoQ and the low Within-laboratory CV. In addition, the results are obtained in 35-minute. We expect this novel immunoassay could reduce the burden on operators and turnaround time.

B-271 High Sensitivity Homogeneous Enzyme Immunoassay for Benzodiazepines and Metabolites

Abstract Background Benzodiazepines are widely prescribed for treating various conditions such as anxiety, insomnia, and seizures due to their sedative and anxiolytic properties. Despite their therapeutic benefits, concerns persist regarding their potential for misuse, dependence, and addiction. Urine drug screening immunoassays serve as an important tool in monitoring benzodiazepine usage, ensuring adherence to prescribed regimens, identifying misuse or abuse, and facilitating necessary medical interventions. However, limitations of benzodiazepine immunoassays are well-documented in literature, with issues surrounding their lack of sensitivity to glucuronidated and 7-amino metabolites, inability to detect newer benzodiazepines, and poor cross-reactivity with different benzodiazepines. ARK Diagnostics has developed a highly sensitive homogeneous enzyme immunoassay capable of detecting both classic and designer benzodiazepines, in addition to their glucuronide and 7-amino metabolites in human urine at a cutoff concentration 200 ng/mL, without the need for glucuronidase or sample pretreatment. Methods The ARKTM Benzodiazepine Plus Assay is a liquid-stable homogenous enzyme immunoassay consisting of two reagents. The assay uses temazepam as the 200 ng/mL cutoff calibrator. The performance characteristics of this assay, including precision, spiked recovery, specificity, and method comparison to LC-MS/MS, were evaluated on the Beckman Coulter AU680 automated clinical analyzer. Results The ARKTM Benzodiazepine Plus demonstrated acceptable precision, with no overlap between cutoff (200 ng/mL) and ±25% control levels (150 ng/mL and 250 ng/mL) in a histogram overlap analysis, and ≤3.6% CV in semi-quantitative mode. Spiked temazepam samples spanning the semi-quantitative assay range of 1000 ng/mL were recovered between 95.0% and 103.7% of the spiked levels. Thirty-eight benzodiazepines produced cross-reactivities >80%, including the following benzodiazepines and metabolites: lorazepam, clonazepam, diazepam, alprazolam, temazepam, oxazepam glucuronide, lorazepam glucuronide, temazepam glucuronide, 7-aminoclonazepam, and 7-aminoflunitrazepam. In method comparison studies, a total of 103 urine samples containing benzodiazepines with LC-MS/MS values ranging from 1.3 ng/mL to 3257.6 ng/mL were tested with the ARKTM Benzodiazepine Plus Assay. Eighty samples tested positive with values ≥200 ng/mL and 23 samples tested negative with values <200 ng/mL by the ARKTM Benzodiazepine Plus assay. Of the 23 samples that tested negative, 21 had LC-MS/MS values <200 ng/mL and 2 samples were identified as midazolam samples with metabolite a-OH-midazolam levels >200 ng/mL. Conclusions The ARKTM Benzodiazepine Plus Assay enables the sensitive, rapid, and reliable measurement of benzodiazepines and their metabolites in human urine, without the need for hydrolysis or pretreatment. The assay addresses the present constraints of currently available benzodiazepine immunoassays, including their insufficient sensitivity, limited cross-reactivity to metabolites, and the requirement for glucuronidase pretreatment. The ARKTM Benzodiazepine Plus Assay is applicable to a wide range of clinical chemistry analyzers.

B-087 Sars-cov-2 Nt Chip test performance: a novel microarray-based enzymatic immunoassay for in vitro identification of neutralizing antibodies against emerging variants

Abstract Background The COVID-19 pandemic has created a need for reliable and adaptable diagnostic tools to detect exposure and neutralizing antibodies to emerging SARS-CoV-2 variants. To address this need, we evaluated the performance of the SARS-CoV-2 NT Chip Test, an ELISA-like immunoblot assay that incorporates a customizable panel of purified antigens from the variants of interest. This assay detects antibodies that neutralize the interaction between the receptor-binding domain (RBD) of the Wuhan strain and the Omicron BA1 variant with the cellular ACE-2 receptor and provides a marker of prior infection through the nucleocapsid protein. Methods We analyzed 73 clinical samples from six distinct groups: GROUP 1: Pre-pandemic samples (33); GROUP 2: Samples from children naive to Wuhan and infected with the Omicron variant (10); GROUP 3: Samples from individuals infected only with the original Wuhan strain (10); GROUP 4: Samples from individuals vaccinated and/or infected with the Wuhan strain and the Omicron variant (10); GROUP 5: Samples collected from individuals with 0-2 days after Omicron BA.1 infection (5) and GROUP 6: Samples collected from the same individuals 10 days after Omicron BA.1 infection (5). Evaluation utilized a proprietary non-numerical (qualitative) validation spreadsheet. Performance metrics included sensitivity, specificity, observed agreement (≥90%), and kappa index (≥0.70). Results were compared against the manufacturer's validation using the PRNT viral neutralization assay. Results Our comparative evaluation showed the following results for each parameter: 1) Wuhan neutralizing antibodies (sensitivity and specificity of 92%, agreement of 92%, and kappa index of 0.84); 2) Omicron neutralizing antibodies (sensitivity and specificity of 100%, agreement of 100%, and kappa index of 1.00); 3) Anti-nucleocapsid antibodies (sensitivity of 97%, specificity of 98%, agreement of 97%, and kappa index of 0.94). Three samples from GROUP 1 showed positive results for Wuhan RBD and one for Nucleocapsid, all close to the cutoff value. Four samples from GROUP 2 showed positive results for Wuhan RBD, which may be attributed to maternal antibodies. Three samples from GROUP 3 and three samples from GROUP 4, showed negative results for nucleocapsid, agreeing with the manufacturer's validation. Samples from GROUP 5 and GROUP 6 showed expected results, with Omicron RDB negative in GROUP 5, becoming positive in GROUP 6. Conclusions The test showed excellent performance in the verification of the three parameters in the populations studied, being approved in our laboratory. Due to its ability to incorporate antigens from emerging variants, it enables sensitive tracking of changes in neutralizing antibody profiles over time. The automated and customizable nature of the test provides an important tool for both public health epidemiological studies, monitoring changes in humoral immunity as this virus evolves, and for individual diagnostics, revealing the level of humoral protection of a patient against circulating strains in a timely manner to guide care. Although further studies are still warranted, the assay's ability to map evolving strain-specific serological immunity may offer valuable insights.

Neuroprotective Actions of Hydrogen Sulfide-Releasing Compounds in Isolated Bovine Retinae

Background: We have evidence that hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H2S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H2S-releasing compounds against hydrogen peroxide (H2O2)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H2S biosynthesis called L-cysteine, with the fast H2S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H2S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H2O2 (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H2S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-β-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α–ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H2S-releasing compounds. Results: Exposure of the isolated retinas to H2O2 produced a time-dependent (10–40 min) and concentration-dependent (30–300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM–1 µM) and NaHS (30 –100 µM) significantly (p < 0.001; n = 12) prevented H2O2-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM–1 µM) attenuated oxidative H2O2 damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H2O2 alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H2O2-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H2S-releasing compounds and the substrate, L-cysteine, can prevent H2O2-induced lipid peroxidation in an isolated bovine retina.

Synthesis and Molecular Docking Studies of New Ibuprofen Derivatives as AChE, BChE, and COX‐2 Inhibitors

AbstractAlzheimer's disease (AD), the most common age‐related neurodegenerative condition, is named after Alois Alzheimer and is marked by a progressive deterioration in memory, cognitive function, and behavior. Research has highlighted the importance of nonsteroidal anti‐inflammatory drugs (NSAIDs) in inhibiting the aggregation of amyloid β‐peptide (Aβ), a key feature of AD pathology. Ibuprofen, an NSAID from the propionic acid class, is widely used to manage osteoarthritis and rheumatoid arthritis, exhibiting strong anti‐inflammatory and antipyretic effects. However, the drug's acidic group limits its selectivity for cyclooxygenase (COX) enzymes and contributes to several adverse effects. This study aimed to modify the acidic moiety of ibuprofen into lactone (IBU‐O 1–4) and lactam (IBU‐I 1–3) derivatives to mitigate these side effects. The structural properties of the synthesized imidazolone (IBU‐I 1–3) and oxazolone (IBU‐O 1–4) derivatives were characterized through Q‐TOF LC‐MS, ¹H‐NMR, ¹3C‐NMR, and IR spectroscopy. Molecular docking studies followed by Ellman's method assessed the inhibitory effects of these compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), while an enzyme immunoassay (EIA) kit was used to evaluate their inhibition of cyclooxygenase‐2 (COX‐2).

Seropositivity and coinfection of hepatitis B and hepatitis C viruses in Central India: A hospital-based study

ABSTRACT Background: Hepatitis B virus (HBV) and Hepatitis C virus (HCV) show similarity in the transmission, distribution, hepatotropism, and leading to chronic asymptomatic infection. Coinfection of HBV and HCV can lead to more severe liver disease and an increased risk for progression to hepatocellular carcinoma (HCC). Most of the people with chronic infection are unaware of their HBV and HCV infections, hence facilitating these to go undiagnosed until these viruses have caused serious liver damage and they act as a potential source of infection for the community at large. Therefore, the present study aimed to find the prevalence of HBV and HCV along with incidences of coinfection of HBV and HCV in patients seeking hospital care in central India. Methods: A five-year hospital-based study was carried out at the tertiary care hospital in Central India from 2018 to 2022. A total of 72402 patients attending the outdoor patients and admitted indoor patients who were advised for HBV and HCV for screening before any invasive/surgical procedure and patients who presented with symptoms of acute or chronic liver disease were included in this study. Screening was done by immunochromatography-based card test followed by the confirmation of all samples by enzyme immunoassay. Results: Seroprevalence of HBV and HCV was found to be 3.71% and 1.91%, respectively. Coinfection with HBV/HCV was seen in 0.13% of the individuals. The overall prevalence of HBV, HCV, and HBV-HCV coinfection was significantly higher in the male population as compared to females. Conclusion: The study findings of seroprevalence of HBV and HCV among the hospital-based population will help to get a baseline understanding of the disease burden in central India. The HBV/HCV coinfection rate also raises serious concerns owing to its high prevalence rate among the younger age.

Analytic Performance of MicroVue Enzyme Immunoassay (EIA) for Measurement of Ba and sC5b-9 for Urine

Analytic Performance of MicroVue Enzyme Immunoassay (EIA) for Measurement of Ba and sC5b-9 for Urine

Comparative Analysis of The Effectiveness of Antihypertensive Therapy Including Spironolactone and Eplerenone in Patients With Essential Hypertension and Atrial Fibrillation.

To perform a comparative analysis of the efficacy of antihypertensive therapy (AHT) containing spironolactone or eplerenone in patients with essential arterial hypertension (AH) and atrial fibrillation (AF). The study included 99 male and female patients with essential AH complicated by permanent AF, who were receiving the outpatient treatment at the National Specialized Scientific and Practical Medical Center of Cardiology (Tashkent). The patients aged 61.3±9.5 years, the mean duration of AH was 12.9±8.3 years. All patients were divided into two groups: Group 1, patients who completed a 6-month combination AHT containing spironolactone (n=51); Group 2, patients who completed a 6-month combination AHT containing eplerenone (n=48). AF was diagnosed by electrocardiogram (ECG) and/or 24-hour ECG monitoring according to standard diagnostic criteria. The ECG study was performed in compliance with the American Society of Echocardiography Guidelines in M- and B-modes. The degree of structural vascular alterations was determined by the intima-media thickness of the common carotid artery by duplex scanning and microalbuminuria in morning urine. The concentrations of sex hormones were measured by the enzyme immunoassay. The serum concentrations of lipids, glucose, creatinine, and uric acid were measured by the enzymatic method. The glomerular filtration rate (GFR) was calculated with the EPI formula. Results of all studies were considered statistically significant at p<0.05. The proportion of patients who achieved the target diastolic blood pressure (BP) values was significantly greater in the eplerenone-containing treatment group than in the spironolactone-containing treatment group: 87.8% vs. 67.5% (p=0.043). The proportion of patients who simultaneously achieved the target systolic and diastolic BP values was slightly greater in the eplerenone-containing treatment group than in the spironolactone-containing group (100% vs. 92.1%, p=0.060). The best cardioprotective efficacy was observed in the group of combination AHT containing eplerenone. Specifically, in Group 2, the left ventricular ejection fraction (LVEF) was significantly improved compared to Group 1: from 55.4±10.6% at baseline to 52.6±9.1% in Group 1 (p>0.05) and from 54.8±8.8% at baseline to 58.2±6.4% in Group 2 (p<0.02). Only in Group 2, the left atrial volume index (LAVI) was significantly decreased compared to Group 1. Thus, in Group 1, the LAVI changed from 42.2±15.1 ml/m2 at baseline to 40.4±12.2 ml/m2 (p>0.05) and in Group 2, from 41.2±15.3 ml/m2 at baseline to 37.3±13.5 ml/m2 after the treatment (p<0.05); the ∆% LAVI in the eplerenone group was -5.9% vs. -0.36% in the spironolactone group. In men of Group 1, estradiol significantly increased from 13.9±12.6 pmol/l at baseline to 22.7±12.4 pmol/l (p<0.001). The good antihypertensive efficacy of the 6-month combination therapy containing eplerenone was significantly superior to spironolactone in achieving the target BP values. The eplerenone-containing treatment significantly improved LVEF and decreased LAVI compared to the spironolactone-containing treatment. A trend towards a beneficial effect of the AHT containing eplerenone on concentrations of sex hormones was noted in both women and men.

Correlation of Serum IL-1β Level in Rheumatoid Arthritis Patients with Disease Severity Parameters

Rheumatoid arthritis (RA) is a systemic inflammatory, chronic disorder, characterized by prolonged inflammation of the synovial joints, with ultimate destruction of joints, high concentrations of IL-1β in the synovial fluid and serum RA patients correlated with RA incidence. This study was conducted to compare IL-1β concentration in serum from patients with RA and healthy controls with the correlation parameter being related to markers indicating disease severity. A case-control study enrolled 71 patients with RA and 46 healthy controls from an Iraqi Arab population. In the present study, the level of serum IL-1β was detected using a quantitative sandwich enzyme immunoassay method of Enzyme-Linked Immunosorbent Assay (ELISA). This study found a correlation between the serum level of IL-1β and DAS28-CRP severity among RA patients (p=0.065). Furthermore, the serum level of IL-1β was significantly increased in RA patients compared to the level noted in the healthy group (p=0.039). Moreover, the results demonstrated a very weak correlation between the IL-1β level with CRP, ESR, and ACCP (r=0.079, r=0.059, r=0.080), respectively. In conclusion, the current study showed a correlation between the serum level of IL-1β and RA disease severity (DAS28) among the Iraqi Arab population, additionally, the study noted a very weak correlation between the IL-1β level with CRP, ESR, and ACCP.

Распространение токсигенных цианобактерий в водных объектах на территории Восточно-Европейской равнины

We analyze the distribution of potentially toxigenic cyanobacteria in water bodies and rivers of the East European (Russian) Plain on the basis of relevant literature and the results of our studies. Special attention is paid to cyanotoxins: microcystins, which are hepatotoxins, cylindrospermopsin, which is hepatotoxin and cytotoxin, and anatoxins and saxi-toxins, which are neurotoxins. Microcystis and Dolichospermum are the most represented genera of potentially toxigenic cyanobacteria, as well as Aphanizomenon flos-aquae Ralfs ex Bornet & Flahault and Planktothrix agardhii (Gomont) Anagnostidis & Komárek species. The above are the most commonly found in water bodies of the study area. The presence of potentially toxigenic cyanobacteria in water bodies was confirmed by the results of phytoplankton studies using molecular genetic methods. The quantitative content of the dissolved in water and intracellular cyanotoxins was determined using enzyme immunoassay methods, high-performance liquid chromatography, and mass spectrometry. It was noted that microcystin- and anatoxin-a-producing cyanobacteria are the most common in most of the studied water bodies and streams, while saxitoxin- and cylindrospermopsin-producing cyanobacteria are less common. The total content of the dissolved microcystins in different water bodies varies from trace amounts up to 1670 μg/L with SanPiN 1.2.3685-21 standard for microcystin-LR 1 μg/L. The maximum recorded anatoxin-a content was 0.600 μg/L. The preparation of this review made it possible to supplement and summarize information on the distribution of toxigenic cyanobacteria and cyanotoxins in rivers, lakes and reservoirs of temperate latitudes, as well as on the quantitative content of cyanotoxins in water.

The Effect of Selenase on Inflammatory and Cytoprotective Markers in Experimental Chronic Generalized Periodontitis

Epidemiological studies in recent decades have revealed a significant increase in the number of patients with periodontal diseases leading to tooth loss. Modern realities require improvement of drug treatment of periodontitis. The antioxidant Selenase, selenium derivative, is an interesting treatment strategy for periodontitis. The study was carried out with the aim to evaluate the healing effectiveness of Selenase in rats with chronic generalized periodontitis (CGP) by its effect on markers of inflammation and cytoprotection. Experimental CGP was modulated in Wistar rats by a calcium-deficient diet with the inclusion of a prooxidant. Selenase (50 mcg/kg) and Mexidol (ethylmethylhydroxypyridine succinate, 250 mg/kg) were administered intragastrically for 30 days. Levels of IL-1β, HIF-1α, HSP70, and TNF-α were determined in the blood after treatment using the enzyme immunoassay method. Experimental CGP was characterized by the development of hyperemia, swelling, and bleeding of the gums; mobility of teeth; and gingival pockets up to 8 mm against the background of increased inflammatory markers (IL-1β, TNF-α), and molecular markers of cytoprotection (HIF-1α, HSP70) in the blood, indicating a homeostatic response of the periodontium in response to inflammation and subsequent hypoxia. Administration of Selenase to rats with CGP produced pronounced healing effects: the reduction in the depth of periodontal pockets by 42.55 %, cessation of bleeding, and disappearance of swelling against the background of a decrease of inflammatory markers: IL-1β – by 44.6 %, and TNF-α – by 65.9 % (p < 0.05). HIF-1α increased by 36.8 %, and HSP70 – by 71.1 % compared to those of the control group, which was not given the treatment (p < 0.05). The results obtained suggest a significant influence of Selenase on HSP70-dependent mechanisms of endogenous cytoprotection. The results of the study found that the use of Selenase in experimental CGP is more effective than Mexidol.

Structural Manipulation of Microwell for Enhancing Analytical Performance of Enzyme Immunoassay

Structural Manipulation of Microwell for Enhancing Analytical Performance of Enzyme Immunoassay

The impact of chronic stress on the hormonal state of women affected by hostilities and displaced women

During times of war, refugee women often have high levels of chronic stress due to factors such as war, displacement and uncertainty. The study focuses on examining the hormonal responses of female refugees experiencing chronic stress from war and displacement.The objective: to assess the impact of chronic stress caused by war and displacement on the hormonal balance of refugee women.Materials and methods. 60 women were examined, who were divided into groups: the 1st group included women who were affected by hostilities (20 persons), and the 2nd group included displaced women (20 individuals). In these 40 women the hormonal studies were conducted in the conditions of military aggression in Ukraine. The control group (3rd group) included healthy women who were examined in the pre-war period. All examined women were 20–25 years old with an average body weight of 55–60 kg. Concentrations of progesterone, estradiol, follicle-stimulating hormone (FSH) and luteinizing hormone, prolactin in the I and II phases of the menstrual cycle (MC), cortisol in the I phase of the MC were determined in blood serum using the enzyme immunoassay method. An analysis of the results of research on the role of the hypothalamic-pituitary-adrenal system in the regulation of the stress response was carried out.Results. In the examined women, various disorders of MC were observed, such as: menometrorrhagia, hypermenorrhea, acyclic uterine bleeding, oligomenorrhea, and amenorrhea. The most pronounced changes in menstrual function were observed in women who were affected by hostilities. Such significant disorders of MC in women of this group coincided with changes in indicators of gonadotropic hormones of the pituitary gland and sex hormones in the blood.A study of the psycho-emotional state of women who suffered from hostilities established that these women have a significant stressful effect of various risk factors on the body. Changes in the levels of cortisol, prolactin and other hormones were detected.Conclusions. Studies have shown that chronic stress affects the hormonal status of women affected by hostilities and of displaced women, including FSH, estradiol, and prolactin levels.A significant increase in the level of FSH in the follicular phase of the menstrual cycle and an increase in the level of estradiol in the luteal phase were determined, which may indicate a compensatory reaction of the body to stress. A significant increase in the level of prolactin was also found, which can cause disorders of menstrual function and affect the psycho-emotional state.

Seasonal and Sexual Variations in Corticosterone and Total Triiodothyronine: A Pilot Study in Mediterranean Tortoises (Testudo hermanni).

The Mediterranean tortoise Testudo hermanni inhabits different regions bordering the northwestern Mediterranean. This species is vulnerable, protected by legislation, and involved in various breeding and reintroduction programs. Wild populations face numerous environmental and anthropogenic stressors that can potentially interfere with their conservation. While seasonal changes in stress-response biomarkers, such as glucocorticoids and thyroid hormones, have been widely studised in mammals and birds, there is a paucity of research in reptile species. Therefore, the present study aimed to evaluate the seasonal fluctuations in corticosterone and total triiodothyronine levels in adult and juvenile Hermann's tortoises (Testudo hermanni) as a measure of the physiological stress response. Blood samples were collected seasonally (winter, spring, summer, and autumn) and posteriorly analyzed by using a specific and validated enzyme immunoassay for both hormones, respectively. The results showed that corticosterone levels varied seasonally and differed between sexes, whereas total triiodothyronine levels changed seasonally but did not differ between sexes. Notably, juveniles exhibited no seasonal changes in either corticosterone or total triiodothyronine levels. Additionally, no correlation between blood extraction duration and hormonal concentrations was observed. This study is pioneering in its comprehensive evaluation of corticosterone and total triiodothyronine changes across all four seasons, including winter, and its focus on juvenile Hermann's tortoises.

Tryptase levels in children with cutaneous mastocytosis: a prospective study

BACKGROUND: Serum tryptase levels are used as a diagnostic marker in mastocytosis and are considered an indicator of clonal mast cell (MC) load. AIMS: To analyze tryptase levels in children with different clinical types of cutaneous mastocytosis. MATERIALS AND METHODS:A single-center prospective study was conducted for the period 2022-2024. The results of examination of 202 children aged from 6 months to 17 years with a diagnosis of cutaneous mastocytosis, who were undergoing outpatient treatment at the Moscow scientific and practical Center of dermatovenereology and cosmetology, were analyzed. Serum tryptase levels were determined by enzyme immunoassay. RESULTS:The median basal tryptase level for the entire group (n=202) was 5.09 μg/L (range 1.3–57.0 μg/L). Serum tryptase values exceeding 11.0 μg/L were detected in 17.3% of patients, and 20.0 μg/L in 7.4%. The mean enzyme level in the control group was 2.55 μg/L. There were significant differences in tryptase concentrations between the maculopapular cutaneous mastocytosis (MPCM) and control groups (p=0.05). The median tryptase level in children with MPCM was 8.7 μg/L. The most pronounced differences were observed between the enzyme levels in children with the monomorphic type of MPCM and in the control group (p=0.01). Analysis of tryptase levels depending on gender and clinical type of MPCM showed that children and adolescents with the monomorphic type of MPCM had higher enzyme levels. CONCLUSIONS: Further studies are needed to determine which tryptase levels in children are indicative of MC activation and primary MC diseases. A detailed understanding of tryptase levels in children may facilitate the development of new specific therapeutic approaches to the management of various clinical forms of cutaneous mastocytosis.