Fluorescence Immunoassay Method Research Articles

Establishment of Time-Resolved Fluorescence Immunoassay for Thyrotropin Receptor Antibodies and Clinical Application.

Thyroid-stimulating hormone receptor antibody (TRAb) is a specific marker for Graves' disease (GD) and the measurement of which can improve the accuracy of GD diagnosis. Current detection methods utilize porcine-derived polyclonal-TRAb, which is unstable and is a source of significant inter-assay variability. This study aims to establish a time-resolved fluorescence immunoassay (TRFIA) method based on stable source of recombinant human TSHR and TRAb for the detection of serum TRAb. The neutralization inhibition method was used in this study. The specific binding of goat-anti-mouse IgG and chimeric construct Fc fragment human TSHR were immobilized on the microplate, and serum TRAb was detected by the competition between Eu3+-labeled human TRAb and the serum TRAb. The TRAb-TRFIA has a wide linear range (0.081-50 IU/L). The intra-assay precision was 2.17-5.41% (< 10%), and the inter-assay precision was 5.75-9.58% (< 15%). No cross-reactivity was observed between TRAb and recombinant human IgG1, IgG2, IgG3, or IgG4. The recovery rate was 106.37%. The TRAb-TRFIA method was significantly correlated with the Roche chemiluminescence method (R2 = 0.9130). TRFIA based on recombinant human TSHR and TRAb with high stability, strong specificity, high sensitivity and wide linear range was established for the detection of TRAb, which can be used for clinical detection.

Urine PLA2R Antibody Detection in Hazard Stratification of PLA2R-Associated Membranous Nephropathy.

M-type phospholipase A2 receptor (PLA2R) is the major autoantigen of membranous nephropathy (MN). As the specific antibodies of MN, the correlation between serum PLA2R antibody (sPLA2R-Ab) levels and PLA2R-associated MN (PMN) risk stratification is still controversial. To apply the time-resolved fluorescence immunoassay (TRFIA) method on urine PLA2R-Ab (uPLA2R-Ab), detect, and then establish a more sensitive method of combined serum and urine PLA2R-Ab detection for PMN hazard stratification. A highly sensitive TRFIA method was used to detect the initial serum and urine samples of patients with PMN. Patients were grouped into remission and nonremission groups according to the outcomes after 12 months of treatment and the data were analyzed. The cutoff values of sPLA2R-IgG (sPLA2R-immunoglobulin G), uPLA2R-IgG, sPLA2R-IgG4, and uPLA2R-IgG4 for distinguishing between remission and nonremission groups were 50 relative units (RU)/mL, 3.51 RU/mL, 6835 ng/mL, and 143.4 ng/mL, respectively. The average value in the remission group for sPLA2R-IgG, uPLA2R-IgG, sPLA2R-IgG4, and uPLA2R-IgG4 was 37.39 RU/mL, 1.10 RU/mL, 3498.99 ng/mL, and 33.83 ng/mL, respectively. The average value in the nonremission group for sPLA2R-IgG, uPLA2R-IgG, sPLA2R-IgG4, and uPLA2R-IgG4 was 279.96 RU/mL, 45.36 RU/mL, 25762.47 ng/mL, and 1383.89 ng/mL, respectively. For sPLA2R-Ab as the primary factor, in combination with uPLA2R-Ab, the high-risk predictive value of combined detection of serum and urine PLA2R-IgG and of serum and urine PLA2R-IgG4 was upgraded from 54.55% to 100% and from 75% to 100%, respectively. A highly sensitive TRFIA method was applied in this study; the combined detection of serum and urine PLA2R-Ab improves the efficiency of PMN risk stratification, and can provide a better assessment of PMN monitoring.

Therapeutic drug monitoring of methotrexate by disposable SPCE biosensor for personalized medicine

BackgroundMethotrexate (MTX) is widely used in clinical practice for the treatment of malignant tumors and autoimmune diseases. High-dose MTX has been shown to be an effective approach for treating various malignant tumors, but it is accompanied by numerous toxic side effects, necessitating therapeutic drug monitoring (TDM) for patients and timely “folinic acid rescue.” High-performance liquid chromatography and fluorescent immunoassay (FIA) are currently used to detect MTX, but these methods are limited by complex sample preparation, time consumption, and high cost. Therefore, a simple, rapid, and cost-effective MTX measurement method is required. ResultsWe developed a flexible and inexpensive electrochemical sensor using a stearyl trimethyl ammonium bromide (STAB)-modified, screen-printed carbon electrode to directly detect MTX in human serum. Assay performance was validated via detection of MTX in spiked buffer. The sensor was capable of measuring MTX concentrations ranging from 0.01 to 1 μM and 1–1500 μM, with a limit of detection of 3.1 nM and a limit of quantitation of 3.5 nM. For the samples simulating combined medication, the sensor exhibited outstanding selectivity, in cross-reactivity, with the maximum response value of interferents reaching only 3.49 %. Additionally, the sensor shows reliable repeatability with a relative standard deviation of 3.8 % and remarkable stability. Recovery in human serum validated the clinical utility of the sensor in point-of-care testing conditions. The sensor's applicability to personal medicine was confirmed by detecting MTX blood concentration in patients with different diseases. The results obtained by the sensor were compared with those obtained by the FIA technique, a method commonly used in hospitals, showing a high level of consistency between both methods. SignificanceTo meet the requirements of personalized medicine for MTX-patients, we developed a disposable biosensor with wide detection range from 0.01 to 1 μM and 1–1500 μM. Owing to the effective enrichment of STAB, the electrochemical response was sensitive, selective, stable, and rapid. As per the clinical test results, our sensor has shown a high level of consistency with the FIA method, indicating its potential to replace FIA as a cost-effective platform for MTX-TDM.

Establishment of a high-sensitivity time-resolved fluorescence immunoassay with PLA2R-IgG1 antibody and its clinical application in idiopathic membranous nephropathy prognosis

IntroductionThe objective of this study was to develop a highly sensitive time-resolved fluorescence immunoassay (TRFIA) method to detect phospholipase A2 receptor (PLA2R)-IgG1 antibodies and evaluate its clinical relevance in predicting the prognosis of individuals with idiopathic membranous nephropathy (IMN). Materials and methodsA three-step indirect TRFIA method was established using a PLA2R antigen-coated microtiter plate to capture PLA2R-IgG antibodies, followed by detection using mouse anti-human IgG1 and Eu3+-labeled goat anti-mouse IgG antibodies. This method was applied to the initial serum of 56 patients with PLA2R-IMN to investigate the clinical value of PLA2R-IgG1 antibody levels in predicting IMN prognosis. ResultsThe detection range of PLA2R-IgG1-TRFIA was 0.85–300RU/mL, with intra-assay precision of 3.54–5.93 % and inter-assay precision of 4.39–9.36 %. Recoveries were 101.77–108.04 %. A PLA2R-IgG1 level above 2.21RU/mL indicated PLA2R-IMN. At initial diagnosis, the median PLA2R-IgG level was 51.24RU/mL in the remission group and 93.27RU/mL in the non-remission group. The median PLA2R-IgG1 level was 603.32RU/mL in the non-remission group, which was 4.29 times higher than that in the remission group (140.67RU/mL). PLA2R-IgG1 levels (P = 0.001) more effectively distinguished between remission and non-remission groups compared with PLA2R-IgG levels (P = 0.094). ConclusionsThe first quantitative TRFIA for PLA2R-IgG1 was established, showing greater clinical value in predicting IMN prognosis, compared to that for PLA2R-IgG levels.

CORRELATION BETWEEN BMI AND GONADOTROPIN HORMONES IN THE FEMALE GENDER

Introduction: The pituitary gland affects growth and development. Its main function is in the activity of sexual/reproductive hormones and in the functioning of the glands (thyroid gland, adrenal glands and gonads). Gonadotropin hormones (gonadostimulins) are so named because they stimulate the function of the gonads - male and female sex glands (testicles and ovaries) in which sex cells - sperm and egg cells are created. Overweight is a state of metabolic disorders where excess body fat accumulates to the extent that it can negatively affect human health. Obesity as a BMI factor is related to the functioning of the thyroid gland with its hormones and the pituitary gland with gonadotropin hormones. Purpose of the study: This paper aims to present the correlation between BMI and gonadotropin hormones in reproductive and postmenopausal women. The aim of the paper will also consist in the status of gonadotropin hormones and the functional state of these hormones by comparing them with metabolic disorders such as BMI, obesity and the synthesis of follicle stimulating hormone and gonadotropin hormone. Material and method: A total of 20 patients will be included in the research, divided according to age group into two categories. The first group will include patients aged 10-40 years, the second group patients over 41 years old. Blood (serum) samples will be taken from patients for analyzing gonadotropin hormones: FSH, LH, PRL and thyroid hormones: TSH, FT4, FT3. They will be analyzed with the modern automatic fluorescent enzyme immunoassay method (Vidas-Biomerie). The BMI of the body will be measured with a metric formula where the weight of the patient (in kg) divided by the height of the patient (in m2) set to the square power will give us the measure of body index-BMI (%). SPSS – Software package version 20.0 Numerical series were analyzed using central tendency measures (average, median, minimum values, maximum values), as well as distribution measures (standard deviation); To determine the regularity of the frequency distribution of the examined variables, the Shapiro-Wilk W Test was used; Non-parametric tests for two independent parameters were used to test the significance and difference between some numerical parameters with irregular frequency distribution (Mann Whitney U test); To determine statistical significance, a two-way analysis was used with a significance level (means) of p&lt;0.05. Results: The results of the research will give a real insight into the data of gonadotropic hormones FSH, LH, PRL and thyroid hormones TSH, FT4, FT3 of the female sex in the Tetova region. The results of the research will also provide information about the correlation of BMI and gonadotropin and thyroid hormones. Conclusion: From the analyzes performed on patients of different age groups we come to the conclusion that: • Gonadotropin hormonal disorders can lead to the phenomenon of metabolic diseases such as BMI. • These disorders can have consequences such as: polycystic ovary diseases in women, Hashimoto's genetic syndrome, Graves, infertility in women, etc.

Accelerating the Phosphatase-like Activity of Uio-66-NH2 by Catalytically Inactive Metal Ions and Its Application for Improved Fluorescence Detection of Cardiac Troponin I.

Compared with natural enzymes, nanozymes usually exhibit much lower catalytic activities, which limit the sensitivities of nanozyme-based immunoassays. Herein, several metal ions without enzyme-like activities were engineered onto Uio-66-NH2 nanozyme through postsynthetic modification. The obtained Mn+@Uio-66-NH2 (Mn+ = Zn2+, Cd2+, Co2+, Ca2+and Ni2+) exhibited improved phosphatase-like catalytic activities. In particular, a 12-fold increase in the catalytic efficiency (kcat/Km) of Uio-66-NH2 was observed after the modification with Zn2+. Mechanism investigations indicate that both the amino groups and oxygen-containing functional groups in Uio-66-NH2 are the binding sites of Zn2+, and the modified Zn2+ ions on Uio-66-NH2 serve as the additional catalytic sites for improving the catalytic performance. Furthermore, the highly active Zn2+@Uio-66-NH2 was used as a nanozyme label to develop a fluorescence immunoassay method for the detection of cardiac troponin I (cTnI). Compared with pristine Uio-66-NH2, Zn2+@Uio-66-NH2 can widen the linear range by 1 order of magnitude (from 10 pg/mL-1 μg/mL to 1 pg/mL-1 μg/mL) and also lower the detection limit by 5 times (from 4.7 pg/mL to 0.9 pg/mL).

Differences of Hepatitis B Serological Tests in Cirrhosis and Hepatocellular Carcinoma Patients

Cirrhosis and hepatocellular carcinoma (HCC) are the most common complications of chronic hepatitis B. Hepatitis B Virus (HBV) reactivation occurs in the inactive phase, characterized by reappearance of HBsAg or negative HBsAg. The prevalence of occult hepatitis B infection in cirrhosis and HCC ranges from 20% to 60%. This study aimed to analyze the differences in hepatitis B serological tests in patients with cirrhosis and HCC for diagnosis of acute or chronic hepatitis B. The current research was analytical and descriptive with a cross-sectional study design. This study involved 177 patients, including 50 cirrhosis patients and 127 HCC patients. Hepatitis B serological tests (HBsAg, HBeAg, anti-HBe, total anti-HBc) were analyzed using VIDAS instruments by the Enzyme-Linked Fluorescent Immunoassay (ELFA) method. Hepatitis B serological test results were grouped based on the interpretation of serological test results. The data were processed with the statistical test Kolmogorov-Smirnov test, independent T-test, and Chi-Square, and results with p&lt;0.05 were reported as significant. This study found that most males suffer from cirrhosis and HCC due to chronic HBV infection, with a mean age of 57. Chronic hepatitis was most common in patients with cirrhosis and HCC (71.2%). There was no significant difference in the interpretation of the hepatitis B serological test between patients with cirrhosis and HCC, with a p-value of 0.230 (p &gt; 0.05). There was no significant difference in interpreting the hepatitis B serological test between cirrhosis and HCC. Both reactive HBsAg and non-reactive HBsAg can be obtained in cirrhosis and HCC.

Development of a sensitive fluorescent immunoassay based on fluorescent nanoparticles labeling for the quantitation of fipronil in edible flowers

Edible flowers have long been used for tea, functional foods, and spices, however, during planting, these natural products are vulnerable to pests and fungal diseases. In recent years, the presence of pesticide residues in edible flowers has attracted extensive attention and of the pesticides found in these products, fipronil residue has been widely reported. Therefore, it is imperative to develop a simple, sensitive, and reliable method for detecting fipronil in edible flowers. In this study, a novel labeled antibody was synthesized by using fluorescent nanoparticles as a secondary antibody, and a fluorescence immunoassay method was established with the conjugated conjugate. The limits of detection and half maximal inhibitory concentration of this method were 0.21 μg/L and 8.61 μg/L, respectively. Furthermore, the developed method was 3.5 times more sensitive than the enzyme-linked immunosorbent assay. After verification, the established method was successfully applied to determine fipronil levels in edible flower samples, and then validated with a gas chromatography-tandem mass spectrometry method. To our knowledge, this is the first report on the successful application of fluorescent nanoparticles in the detection of fipronil in edible flowers.

KORELASI KADAR C-REACTIVE PROTEIN DENGAN JUMLAH TROMBOSIT PADA PENDERITA DIABETES MELITUS TIPE 2

Type 2 diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia and insulin resistance, associated with decreased nitric oxide (NO) levels, increased free radical production, inflammation, endothelial dysfunction, and progressive vascular damage. C-reactive protein (CRP), an acute phase protein regulated by pro-inflammatory cytokines like interleukin-6 (IL-6), serves as a systemic inflammation marker. Pentameric CRP (pCRP) and monomeric CRP (mCRP) are implicated in mediating inflammation and endothelial dysfunction, with roles in endothelial cell adhesion, phagocytosis, inflammatory signaling, and interactions with endothelial cells, neutrophils, macrophages, and platelets. Additionally, leukocytes secrete platelet-activating factors, enhancing platelet activation and cytokine and chemokine release. This descriptive analytical study with a cross-sectional design aimed to investigate the correlation between CRP levels and platelet counts in patients with type 2 DM. Utilizing purposive sampling, 47 patients diagnosed with DM at Sultan Syarif Mohamad Alkadrie Hospital, Pontianak, during June 2023 were selected. CRP levels were measured using the fluorescence immunoassay (FIA) method, while platelet counts were determined through the impedance method. Statistical analysis employing Kendall's Tau-b test revealed a significant correlation (p &lt; 0.05, r = -0.508), indicating that elevated CRP levels are significantly associated with reduced platelet counts in type 2 DM patients.

EFFECTS OF DHIKR ON SPIRITUALITY AND CORTISOL LEVELS AMONG PATIENTS WITH HEART FAILURE: A PILOT STUDY

Introduction: Depression and anxiety are common comorbid conditions in patients with heart failure (HF). Spirituality was found to have a positive impact on patients’ mental health status. However, whether the spiritual practice affects physiological modulation is still questionable. Therefore, this study aimed to analyze the effect of dhikr as spiritual practice on spirituality and cortisol levels among patients with HF. Methods: A quasi-experiment was used in this study. A sample size of 18 respondents consists of 9 respondents of the control group and 9 respondents of the treatment group. Dhikr intervention was conducted in three meetings with an interval of two days during patients’ hospitalization. Spirituality was measured using a spirituality questionnaire, whereas the cortisol levels with venous blood sampling, measured by the enzyme-linked fluorescent immunoassay (ELFA) method. Statistics analysis in this study was using t-Test for spirituality, Mann Whitney and Wilcoxon Sign Rank Test for cortisol level with a significance level of α&lt;0.05. Results: The results showed that dhikr improves the spirituality of HF patients (p=0.000) and decreases the level of cortisol (p=0.015) significantly. Dhikr interventions appears to strengthen spirituality in patients with HF by improving stress perception which further modulates the adaptive stress response in the form of decreasing cortisol levels. Conclusions: Dhikr could be one of the spiritual nursing interventions to improve spirituality and stress hormonal modulation in patients with HF.

Associations between Borg's rating of perceived exertion and changes in urinary organic acid metabolites after outdoor weight-bearing hiking.

Developing methods to monitor exercise load and evaluate body fatigue and muscle injury over time in hiking training remains a key problem to be solved. A widely used psycho-physical tool to assess the subjective perception of effort during exercise is Borg's rating of perceived exertion (BRPE) scale. Data on the relationships and validity of the BRPE compared to objectively assessed metabolic criteria are still lacking, especially urinary organic acid concentrations. To verify whether the BRPE scale could be used in the prescription of outdoor hiking with weight-bearing and reveal the relationship between the BRPE scale and urinary physiological measures. Eighty-nine healthy men (average age: 22 years) were enrolled in a 40 km (6 h) hiking training exercise with a 20 kg load. After training, the BRPE scale (6-20) was completed. All participants were divided into three groups according to the rating of the BRPE scale. Urine samples were collected before and after training. Urinary myoglobin levels were measured immediately using the fluorescent immunoassay method. The remaining urine was subpacked and frozen for the subsequent detection of urinary organic acids using gas chromatography and mass spectrometry. The contents of organic acids and myoglobin in urine were significantly increased after participants hiked 40 km (6 h) with a 20 kg load. Only orthogonal partial least-squares discrimination analysis performed well in separating the group with a BRPE score of 6-12 from the group with a BRPE score of 13-20. Significant differences in the urine levels of several organic acids were observed between the two groups, and the heatmap also presented different metabolic profiles based on BRPE. According to the standard of a variable importance in the projection > 1, fold change > 1.5 and P < 0.05, 19 different metabolites of urinary organic acids were screened and enriched in pathways mainly including the citrate cycle (tricarboxylic acid cycle) and alanine, aspartate and glucose metabolism. The BRPE scale identified significantly different urinary organic acid profiles between the higher and lower BRPE value groups, and, thus, could be used to monitor body fatigue in individuals participating in long-distance outdoor hiking with weight bearing.

Association of appetite hormone levels with HbA1c in obese type-2 diabetes mellitus patients

Background: Nesfatin-1 and leptin are hormones that control appetite and affect glycemic control. Nesfatin-1 and leptin levels are affected by body mass index (BMI). Hemoglobin A1c (HbA1c) is an objective parameter to assess long-term glycemic control. This present research aims to know the relationship between levels of nesfatin-1 and leptin with HbA1c values ​​as an indicator of long-term objective glycemic control in patients with T2DM with obesity. Methods: Cross-sectional study on 54 obese T2DM subjects. Measurement of nesfatin-1 and leptin levels using the enzyme linked immunosorbent assay (ELISA) method. The value of HbA1c was measured by the fluorescent immunoassay (FIA) method. The correlation between nesfatin-1 and leptin with Hb1c was tested using the Spearman correlation analysis test (p &lt;0.05 was considered significant). Results: The median value of nesfatin-1 levels was 1,824.0 (134.0-5,255.0) pg/mL, the median value of leptin levels was 14.0 (0.4 - 66.4) ng/mL, and the median value of HbA1c was 7.70 (4.9-15.0)%. There is no correlation between nesfatin-1 levels and HbA1c values (p = 0.282 and r = 0.149). There is no correlation between leptin levels and HbA1c values (p value = 0.895 and r = 0.018). Conclusion: HbA1c levels are not associated with an increased appetite, as evidenced by no association between appetite biomarkers and glycemic control.

Correlation of Triglyceride/HDL-Cholesterol Ratio and Visceral Adiposity Index with 25(OH)D in Obese Female

Obesity is a condition of excess body fat mass with cardiometabolic complications. The levels of serum 25-hydroxyvitamin D (25(OH)D) decrease in obesity because it is stored in adipose tissue compartments and is related to dyslipidemia conditions. A high triglyceride/HDL-cholesterol (TG/HDL-C) ratio in obesity is related to dyslipidemia. Visceral Adiposity Index (VAI) is one of the parameters indicating central obesity related to visceral fat distribution in dyslipidemia conditions. This study aimed to prove a correlation between TG/HDL-C ratio and VAI with 25(OH)D levels in obese females. Observational study with cross-sectional design in 66 female patients. HDL-C examinations and triglyceride using the enzymatic colorimetry method. The TG/HDL-C ratio was calculated by dividing TG by HDL. The equation obtained visceral adiposity index (WC/(36.58+(1.89xBMI)) x (TG/0.81) x (1.52/HDL). 25(OH)D examinations used the Enzyme Linked Fluorescent Immunoassay (ELFA) method. Relationship status used the spearman rank test (p&lt;0.05). Median 25(OH) levels were 9.75 (8-18.6)ng/mL. There was a weak negative correlation between TG/HDL-C ratio with 25(OH)D levels in obese females (p=0.020; r= -0.287) and VAI with 25(OH)D in obese females (p=0.019; r= 0.287). There was a weak negative correlation between TG/HDL ratio and VAI with 25(OH)D in obese female patients.

Utilization of a Novel Immunofluorescence Instrument Prototype for the Determination of the Herbicide Glyphosate.

An enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the quantitative analytical determination of the herbicide active ingredient glyphosate in environmental matrices (surface water, soil, and plant tissues). Glyphosate, as a ubiquitous agricultural pollutant, is a xenobiotic substance with exposure in aquatic and terrestrial ecosystems due its extremely high worldwide application rate. The immunoassay developed in Project Aquafluosense is part of a fluorescence-based instrumentation setup for the in situ determination of several characteristic water quality parameters. The 96-well microplate-based competitive immunoassay method applies fluorescence signal detection in the concentration range of 0–100 ng/mL glyphosate. Application of the fluorescent signal provides a limit of detection of 0.09 ng/mL, which is 2.5-fold lower than that obtained with a visual absorbance signal. Beside the improved limit of detection, determination by fluorescence provided a wider and steeper dynamic range for glyphosate detection. No matrix effect appeared for the undiluted surface water samples, while plant tissues and soil samples required dilution rates of 1:10 and 1:100, respectively. No cross-reaction was determined with the main metabolite of glyphosate, N-aminomethylphosphonic acid, and related compounds.

Quantitative detection of malachite green in sediment by a time-resolved immunofluorescence method combined with a portable 3D printing equipment platform

Rapid detection technology of aquaculture fishery drug residues is needed to supplement large-scale instrument methods. To do this, the time-resolved fluorescence immunoassay (TRFIA) method and portable three-dimensional (3D) printing equipment platform were used, in combination with smartphones, to detect malachite green (MG) in pond sediments. The TRFIA was coupled to MG monoclonal antibodies (mAb) through lanthanide metal microspheres europium (Eu3+). The labeled antibody produced competitive immunity in the immune reaction system, and the specific fluorescence intensity in the product was determined by a portable 3D printing equipment platform to achieve quantitative analysis. To test this method, leucomalachite green (LMG) was converted to MG by oxidation of dicyanoquinone (DDQ), and a qualitative analysis was achieved. Methodological evaluation results were satisfactory, recoveries were 83 %–104 %, the limit of detection (LOD) was 0.3 ng/g, the limit of quantitation (LOQ) was 0.7 ng/g, and the coefficient of variation was 1.3 %–7.3 %. The linear equation y = −0.1496x + 0.5585 was in the range of 0–10 ng/g. The linear regression correlation coefficient was 99.2 %. The TRFIA was confirmed and positive samples were measured. Results were consistent with the standard method, which demonstrated that the TRFIA was feasible and that the detection results were reliable. Compared with the national standard method, the TRFIA saves time, is more convenient, and has high sensitivity. It provides an efficient technical method for the rapid screening of MG in the sediments of aquaculture environments.

Feline Herpesvirüs Tip-1 İle Enfekte Kedilerde Vitamin D Düzeyleri

In this study, it was aimed to measure vitamin D levels in Feline Herpesvirus Type-1 (FeHV-1) infected cats and thus to determine its relationship with the disease activity. The animal material of this study was composed of 20 cats of different breeds, ages and genders, which showed clinical symptoms of the disease and were diagnosed with Feline Herpesvirus infection by rapid test. The control group of the study consisted of 10 cats of different breeds, ages and genders, which were healthy according to the results of routine physical examination, laboratory works, which were brought for the purpose of vaccination, and which were determined to be Feline Herpesvirus Type-1 antigen negative with the rapid test. Blood samples were taken from cats in both groups and 25-hydroxy vitamin D3 levels were determined by the fluorescent immunoassay method. The mean standard deviation values of 25 hydroxyvitamin D3 levels in FeHV-1 infected cats and healthy cats were found to be 33.30 and 64.70 ng/ml, respectively. FeHV-1 infected cats showed a significant decrease in serum vitamin D levels compared to healthy cats in the control group. As a result, vitamin D deficiency may have an effect on the formation of the disease.

Current Status of Newborn Screening for the early Diagnosis of Congenital Hypothyroidism in Bangladesh

Background: Newborn screening programs signify early and presymptomatic detection of treatable disorders which authorize commencement of prompt medication to alleviate notable fatality. Newborn screening (NBS) for congenital hypothyroidism (CH) has been a successful public health initiative in preventive medicine over the last few decades. Previous pilot (1999-2006) and phase 1 (2006-2011) NBS program in Bangladesh reported an increased incidence compared to global data which urged to initiate a second phase NBS program for the detection of CH. Materials &amp; Methods: Neonates’ blood was collected from the umbilical cord or by heel prick and drawn on the Guthrie filter paper card. Thyroid-stimulating hormone (TSH) was measured from this filter paper by Immunoradiometric assay (IRMA) or Dissociation Enhanced Lanthanide Fluorescence Immunoassay (DELFIA) method. Results: 123 out of 2,61,550 neonates have already been diagnosed with congenital hypothyroidism from September 2018 to August 2020, which represents an incidence of 1:2126. Conclusion: Ongoing phase-2 project data is already showing some optimistic outcomes. Hence, a time-worthy sustainable policy should be implemented to mark this program as an effective one. Bangladesh J. Nuclear Med. 25(1): 15-21, 2022

Comparison of Diagnostic Tests for Detection of Nonstructural-1(NS1) Antigen Dengue virus using Immunochromatography and Fluorescence Immunoassay Methods

Background : NS1 is currently widely used for diagnosis of dengue virus (DENV) infection. Various methods are used to diagnose DENV infection (DVI), either ELISA, immunochromatography (ICT) or most recently the fluorescence immunoassay (FIA) method which are commercially available. Objective: This study aimed to compare the detection capabilities of dengue NS1 antigens using (1) Dengue NS1 ICT Ag (Standard Q - SD Biosensor, Inc.), (2) Dengue NS1 ICT Ag (SD Bioline - Standard Diagnostic, Inc), and (3) Dengue NS1 Ag FIA (Standard F - SD Biosensor, Inc.) Methods: This study consisted of serum samples (n=80) with the number of DVI patients (n=50), non-DVI (n=30). All samples were examined using all three commercial kits for NS1 antigen testing. All DVI samples showed results of reverse-transcriptase polymerase chain reaction (RT-PCR - SIMPLEXAᵀᴹ Dengue - Focus Diagnostics) and/or positive dengue NS1 (Panbio® Dengue Early ELISA) antigen. Results: Standard F showed the highest sensitivity (82%) compared to Standard Q (74%) and SD Bio line (74%). These three commercial kits had the same specificity 100%. The positive predictive value all of these kits was 100% each. The negative prediction value of Standard F, Standard Q, and SD Bio line were 76.9%, 63.8%, 63.8%, respectively. These three NS1 antigen tests had a good agreement (κ 0.681-0.774). Conclusions: FIA test performance (Standard F SD - Biosensor, Inc.) were a quick and easy examination, showing a higher sensitivity and specificity than ICT for detecting DENV infection. Further research is needed to confirm the diagnosis of primary or secondary infection.

Fluorescence immunoassay based on phage mimotope for nontoxic detection of Zearalenone in maize

AbstractZearalenone (ZEN) is one of the most common mycotoxin contaminants worldwide. In this study, a phage‐based direct competitive fluorescence immunosorbent assay (P‐dcFLISA) was developed for the detection of ZEN. In this P‐dcFLISA, phage mimotope was used to replace chemically synthesized antigens to improve the safety of experiments. Furthermore, ZnCdSe/ZnS (core/shell) quantum dots (QDs) as fluorescent labeling material replaced chromogenic substrate, which are used in traditional ELISA. The 50% inhibition value (IC50) of P‐dcFLISA was 0.301 ng/ml, which was approximately 12‐fold lower than that of phage‐based indirect competitive enzyme‐linked immunosorbent assay (P‐icELISA). The limit of detection (LOD) of P‐dcFLISA was 0.023 ng/ml and the detection range was 0.060–1.531 ng/ml. In the added recovery assay, recovery rate was 93.18–105.52%, with the coefficient variation (CV) ranging from 8.39% to 10.55%. The establishment of P‐icFLISA not only save time, but also avoid the use of mycotoxins in the detection process, providing a safe method for the detection of mycotoxins. In conclusion, phage‐based fluorescence immunoassay method has great potential application in ZEN detection and agricultural monitoring.

Krebs von den Lungen-6 as Disease Severity Marker for COVID-19 Patients: Analytical Verification and Quality Assessment of the Tosoh AIA-360 Compared to Lumipulse G600II.

Background: Krebs von den Lungen-6 (KL-6) has been proposed as a disease severity marker of COVID-19. All research articles reported the KL-6 assay detected through Fujirebio reagents by Lumipulse G600/G1200 instrument. In the present study, KL-6 assay was analysed through Tosoh AIA-360 and compared with analytical results by Lumipulse G600 in a population of COVID-19 patients. Materials and methods: Sixty-four patients (median age, IQR 67 (58–76) years), all hospitalized for COVID-19 interstitial pneumonia at Siena COVID Unit. KL-6 was measured by two methods, chemiluminescence enzyme immunoassay (CLEIA) and fluorescent enzyme immunoassay (FEIA) method by Lumipulse G600 II and AIA 360 systems, respectively. Results: KL-6 concentrations evaluated by Lumipulse G600II were significantly higher in severe than those in non-severe patients (p < 0.0001) as well as evaluating by AIA360 (p < 0.0001). Receiver operating curve (ROC) curve analysis showed that KL-6 concentrations, by Lumipuse G600II, distinguished severe from non-severe COVID-19 patients with an area under the curve (AUC) of 99.8% and the best cut-off value was 448 U/mL. AUROC between severe and non-severe COVID-19 patients using T0 KL-6 concentrations by AIA360 was 97.4% and the best cut-off value was 398 U/mL. According to T0 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 78 ± 174.8; while using T1 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 48 ± 126 (95% limits of agreement −199–295) between the Lumipulse G600 II and the AIA360 systems. Conclusions: In conclusion, our study demonstrated that CLEIA and FEIA methods for serum KL-6 detection are comparable and reliable. KL-6 was confirmed as an easily detectable and effective biomarker to identify severe COVID-19 patients.