Enzyme-linked Immunoassay Method Research Articles

Combination of distance-based paper analytical devices with ionic liquid-based dispersive liquid-liquid microextraction for enzyme-free bilirubin quantification

This article introduces the first demonstration of integrating distance-based paper analytical devices (dPADs) with ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME) for the quantitative detection of bilirubin levels in human samples. In this approach, bilirubin is preconcentrated and extracted into a small droplet of the extraction solvent using IL-DLLME. The resulting solution is then introduced into an economical and straightforward dPAD sensor. Detection relies on a colorimetric reaction between bilirubin and gold ions (Au3+), leading to the in-situ formation of gold nanoparticles (AuNPs) on the paper substrate. The observed red-purple distance length is directly proportional to the bilirubin level. Under optimization, a linear range between 0.25 nM and 5.0 nM (R2 = 0.9979) and a detection limit (LOD) of 250.0 pM were achieved. The assay exhibits notable precision and selectivity without any interference. With its distance readout feature, this method is simpler and more cost-effective, offering greater sensitivity compared to other methods requiring expensive reagents and instruments. Moreover, the technique shows high accuracy and precision for bilirubin quantification in human control samples, with recoveries ranging from 98.33% to 102.00%. No significant difference was observed between our method and the enzyme-linked immunoassay (ELISA) method. By combining a low-cost microfluidic device with a simple microextraction method, this strategy holds great potential as an alternative analytical method for determining bilirubin. It can be expanded for further applications in analytical sensing for point-of-care (POC) diagnostics.

Chronic Unpredictable Mild Stress Affects Weight Changes Through Circadian Cycle Mechanism

Everyday stress is unpredictable and comes in many different forms. The person is constantly exposed to stress because they cannot handle it. Chronic stress results from this illness. Chronic stress can affect changes in the body's standard mechanisms, including body weight modulated by the circadian cycle. Due to the fact that each person's response to stress is unique, stressful situations might result in two different variations in body weight. The purpose of this study is tobserve the effect of the CUMS on weight changes in Rattus novergicus through the circadian cycle mechanism. 16 chronic stress models of the Rattus norergicus were split into control and treatment groups. The chronic stress was provided using the Chronic Unpredictable Mild Stress (CUMS) approach. The control group did not receive any treatment but was still given maintenance such as providing food, drink and measuring their body weight every day. While the treatment group received CUMs for 20 days. Every morning before the stressor exposure, body weight was recorded. And the measurement of corticosterone levels using the Enzyme-Linked Immunoassay (ELISA) method with the Duplo technique. Before termination, blood is taken at the heart. Corticosterone levels varied across the groups (p = 0.032). The study's changes in body weight (p = 0.00) revealed a difference between the control and treatment groups, with the treatment group's weight being -11.15 ± 7.91. Chronic stress can alter how the body normally functions, mainly how the circadian cycle regulates body weight. Since each person's response to stress is unique, stressful situations might result in two different variations in body weight.

Faecal glucocorticoid analysis as indicator of stress and its effect on the reproduction hormones of the female Malayan sun bear (Helarctus malayanus) in captivity.

Faecal glucocorticoid metabolite (FGM) is frequently used as a non-invasive tool to assess adrenocortical activity in animal conservation and welfare studies. However, studies assessing the stress level and its effect on the reproductive hormones of female Malayan sun bear is lacking. This study aims to assess the level of stress of the female Malayan sun bear in captivity and its effect on the female reproductive hormones of the female Malayan sun bears in captivity in Malaysian Borneo. Through FGM analysis via the enzyme-linked immunoassay (ELISA) method, it was found that the bear living in BSBCC has the lowest level of glucocorticoid. as for the relationship between the level of stress and the female reproductive hormones, the level of estrogen which was indicated by estradiol, decreases as the level of FGM increases. In contrast, the level of progesterone increase as the level of FGM increases. It can be concluded that the level of stress in captivity could affect the level of estrogen and progesterone and a high level of progesterone does not always indicate a good reproductive function in female mammals.

Examination of empathy-like behaviour in nicotine-preferring rat lines

Aim Addiction is an important global health issue, impacting also addicts environment and society. Empathy plays crucial role in establishing successful social relationships and is a fundamental component of social life. The aim of this study is to investigate how nicotine preferring (NP) strain and oral forced nicotine administration affects empathy-like behaviour in rats, with gender differences. Materials and Methods Sprague-Dawley NP rats (10 males/10 females) and wild-type control rats (10 males/10 females) were used. Behavioural tests were administered to all rats before and after oral forced nicotine administration. The behavioural tests were completed in the fourth week of nicotine administration. Anxiety levels that could affect empathy-like behaviour were evaluated with open field, elevated plus maze tests and with blood cortisol levels. Oxytocin receptor and arginine vasopressin (AVP) levels, which have been shown to be related to empathy-like behaviour, were examined in the prefrontal cortex and amygdala regions using the enzyme-linked immunoassay method. Results It was observed that males from the NP strain showed less empathy-like behaviour than all other groups, and nicotine administration did not cause a significant change in the results. Higher levels of locomotor activity (LA) were found in control females than in all other groups. Blood nicotine and corticosterone levels were higher in NP rats. No significant differences were found in AVP and oxytocin receptor levels in the prefrontal cortex and amygdala. Conclusions It was found that coming from an addicted strain particularly reduces empathy-like behaviour in males.

Performa Rapid Diagnostik Tes Hepatitis B dengan ELISA sebagai Gold Standar

The ELISA (Enzyme Linked Immunoassay) method has high sensitivity and specificity, qualitatively and quantitatively detecting Hepatitis B Surface Antigen (HBsAg) levels, but the process is long, expensive and requires special expertise, compared to the Rapid Diagnostic Test (RDT) as a rapid test which is more practical and cheaper. The aim of this study was to compare the sensitivity and specificity of the Entebe Rapid Test Diagnostic (ERTD) produced by PT. Hepatika Mataram, with ELISA as the gold standard in detecting HBsAg. The requirement for interpreting sensitivity and specificity results according to World Health Organization (WHO) standards is a minimum of 95%. Testing with a negative panel of 00 samples and a positive panel of 1 samples from Papua in 012 at -80℃ storage. Testing in April 023 at the Molecular Diagnostics and Research Laboratory, Mataram University Hospital. Statistical analysis using MedCal software. ERD HBsAg produced by PT. Hepatika Mataram has a sensitivity of 95.45%, specificity of 99.50%, positive predictive value of 93.59%, negative predictive value of 99.65%, and accuracy of 99.21%.

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<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 > 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.

Aflatoxins and Ochratoxin A in Tea Sold in Lebanon: Effects of Type, Packaging, and Origin.

Tea is among the oldest and most-known beverages around the world, and it has many flavors and types. Tea can be easily contaminated in any of its production steps, especially with mycotoxins that are produced particularly in humid and warm environments. This study aims to examine the level of ochratoxin A (OTA) and total aflatoxin (AF) contamination in black and green tea sold in Lebanon, evaluate its safety compared to international standards, and assess the effect of different variables on the levels of OTA and AFs. For this, the Lebanese market was screened and all tea brands (n = 37; 24 black and 13 green) were collected twice. The Enzyme-Linked Immunoassay (ELISA) method was used to determine OTA and AFs in the samples. AFs and OTA were detected in 28 (75.7%) and 31 (88.6%) samples, respectively. The average of AFs in the positive (above detection limit: 1.75 μg/kg) samples was 2.66 ± 0.15 μg/kg, while the average of OTA in the positive (above detection limit: 1.6 μg/kg) samples was 3.74 ± 0.72 μg/kg. The mean AFs in black and green tea were 2.65 ± 0.55 and 2.54 ± 0.40 μg/kg, respectively, while for OTA, the mean levels were 3.67 ± 0.96 and 3.46 ± 1.09 μg/kg in black and green tea samples, respectively. Four brands (10.8%) contained total aflatoxin levels above the EU limit (4 μg/kg). As for OTA, all samples had OTA levels below the Chinese limit (5 μg/kg). No significant association (p > 0.05) was found between OTA and tea type, level of packaging, country of origin, country of packing, and country of distribution. However, AF contamination was significantly (p < 0.05) higher in unpacked tea, and in brands where the country of origin, packing, and distributor was in Asia. The results showed that the tea brands in Lebanon are relatively safe in terms of AFs and OTA.

Kruppel benzeri faktör 2 polimorfizmlerinin koroner arter hastalığı riski ile ilişkisi

Purpose: Coronary artery disease is defined as complications that develop in proportion to the prevalence of ischemia due to occlusion of the coronary arteries and the resulting cell death. The development of atherosclerosis is significantly influenced by endothelial cell dysfunction. Kruppel-like factor 2, a transcription factor, has been shown to regulate critical biological events in endothelial biology, such as vascular tone, migration, proliferation, vasoreactivity, and angiogenesis. In our study, it was aimed to clarify the relationship between coronary artery disease and Kruppel-like factor 2 protein levels and C1239A polymorphisms.&#x0D; Materials and Methods: 191 individuals who underwent coronary angiography at Mersin University Faculty of Medicine, Department of Cardiology, Health, Research and Application Center were included in the study. Measurements of serum fasting blood glucose, HDL cholesterol, total cholesterol, and triglyceride levels were performed on the AU5800 (Beckman Coulter, United States) autoanalyzer. Serum LDL levels were calculated using the Friedwald equation. Serum Kruppel-like factor 2 protein levels were measured by sandwich enzyme-linked immunoassay method on the Multiskan GO (Thermo Scientific, Finland) device. Kruppel-like factor 2 C1239A variations were detected on the Applied Biosystem VIIA™ 7 Real-Time PCR (Life Technologies Co., United States) device by TaqMan® single nucleotide polymorphism (SNP) genotyping method.&#x0D; Results: Men had a 3.8-fold higher risk of CAD than women. (Odd’s ratio 3.83, 95% Confidence interval 1.98-7.39; p

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.

Association between bacterial vaginosis and 25-Hydroxy vitamin D: a case-control study

IntroductionBacterial vaginosis (BV) is the most common vaginal dysbiosis among women of reproductive age. Micronutrient deficiencies, including vitamin D deficiency, can increase the risk of BV. The findings of previous studies regarding the relationship between vitamin D deficiency and BV were conflicting. Therefore, this study aimed to evaluate the association between BV and serum level of 25-hydroxy vitamin D.Materials and methodsThis case-control study was conducted in Gonabad County in 2021. One hundred and twenty-five confirmed BV cases and 125 controls who were matched based on age and intercourse frequency (maximum difference of two days per week) enrolled in the study. Data collection was performed using a demographic and reproductive data questionnaire and a checklist for recording Whiff test results, serum 25-hydroxy vitamin D level, litmus paper observation, and microscopic findings (clue cells). Serum level of vitamin D was evaluated based on enzyme-linked immunoassay method (Monobind kit) from 0.5 ml venous blood drawn from each participant. The conditional logistic regression model was used to analyze data.ResultsThe BV cases had significantly lower 25-hydroxy vitamin D serum levels than controls. The odds of BV increased with vitamin D deficiency (Adjusted odds ratio (AOR): 4.34, 95% confidence interval (CI): 1.39–13.4, p = 0.011, FDR q-value = 0.051), vitamin D insufficiency (AOR: 3.65, 95% CI: 1.23–10.85; p = 0.020; FDR q-value = 0.053), cigarette/hookah smoking (AOR: 3.65, 95% CI: 1.23, 10.85; p = 0.020; FDR q-value = 0.053) and lower age at first intercourse (AOR: 1.16, 95% CI: 1.05, 1.28; p = 0.004; FDR q-value = 0.048). The odds of BV was 0.80 and 0.78 times lower in participants who had coitus interruptus (AOR: 0.20, 95% CI: 0.06, 0.63; p = 0.006; FDR q-value = 0.048) and condom use (AOR: 0.28, 95% CI: 0.10, 0.79; p = 0.016; FDR q-value = 0.051), respectively, compared to participants who did not use contraceptives.ConclusionThese findings suggested that lower serum vitamin D levels were associated with an increased risk of developing BV. However, further studies are needed to confirm the results of the present study.

Brain damage serum biomarkers induced by COVID-19 in patients from northeast Brazil.

Neurological symptoms have been often reported in COVID-19 disease. In the present study, we evaluated brain damage associated with the increase of serum levels of neurological biomarkers S100B and neuron-specific enolase (NSE) induced by SARS-CoV-2 infection, in a population from Northeastern Brazil. Thirty-six healthy control (G1) individuals and 141 patients with confirmed COVID-19 were enrolled in this study. Positive-COVID-19 patients were divided into two groups according to the severity of illness by the National Institute of Health (NIH) criteria, 76 patients with mild symptoms for COVID-19 and (G2) and 65 with acute respiratory conditions requiring supplemental oxygenation via intensive care unit (ICU) admission (G3). A follow-up study was conducted with 23 patients from G2 14 (D14) and 21 (D21) days after the onset of symptoms. Serum levels of NSE and S100B were measured using the enzyme-linked immunoassay method (ELISA). Results revealed a significant positive association between G3 patients and S100B serum expression (p = 0.0403). The serum levels of NSE were also significantly enhanced in the G3 group compared to the control (p < 0.0001) and G2 group (p < 0.0001). In addition, clinical features such as symptoms and oxygenation status were not correlated with NSE or S100B serum expression. The follow-up study demonstrated a decrease over time (21days) in NSE serum expression (p < 0.0001). These results suggest that brain damage is followed by acute virus exposure, with no long-term effects. Future work examining COVID-19 recovery will shed light on chronic neurological damage of SARS-CoV-2 infection.

Development of an Immunoassay Method for the Sensitive Detection of Histamine and Tryptamine in Foods Based on a CuO@Au Nanoenzyme Label and Molecularly Imprinted Biomimetic Antibody.

In this paper, a novel biomimetic enzyme-linked immunoassay method (BELISA) was successfully established for the detection of histamine and tryptamine, based on catalytically active cupric oxide@gold nanoparticles (CuO@Au NPs) as a marker and a molecularly imprinted polymer (MIP) as the biomimetic antibody. Under optimized conditions, the detection limitations of the BELISA method for histamine and tryptamine were 0.04 mg L-1 and 0.14 mg L-1, respectively. For liquor spiked with histamine and tryptamine, the BELISA method delivered satisfactory recoveries ranging from 89.90% to 115.00%. Furthermore, the levels of histamine and tryptamine in fish, soy sauce, and rice vinegar samples were detected by the BELISA method and a high performance liquid chromatography method, with no significant difference between the two methods being found. Although the catalytic activity of nanozymes is still lower than that of natural enzymes, the BELISA method could still sensitively determine the histamine and tryptamine levels in food samples.

Prevalence of cardiovascular diseases in kidney transplant recipients and its relationship with asymmetric dimethylarginine, fibroblast growth factor-23 and multiple inflammatory markers.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase, a marker of endothelial damage and progression of atherosclerosis. Research confirms the association of ADMA with an increased risk of cardiac complications and an increased risk of death, graft loss among kidney transplant recipients (KTRs). The aim of our study was to establish the significance of ADMA and FGF-23 as biomarkers of cardiovascular risk as well as predictors of graft failure and progression of chronic transplant kidney disease in comparison to CKD subjects. In addition, an analysis of the relationship between ADMA, FGF23 and cardiovascular diseases in CKD subjects and KTRs was performed. The study group included 132 KTRs. The control group consisted of age- and sex-adjusted 40 individuals with clinically stable CKD. ADMA, FGF-23, hs-CRP and IL-6 were measured by the enzyme-linked immunoassay method (ELISA). Parameters of body mass composition such as fat mass, FTI, lean tissue mass, LTI, body water and overhydration were assessed by multi-frequency bioimpedance analysis (BIA). Cardiovascular diseases (CVDs) were present in 31.8% of KTRs. Independent variables related to nutritional status (SGA, s-albumin), according to multivariate regression, may have an impact on the prevalence of CVD in the kidney transplant recipients' group. Our study findings suggested a correlation between ADMA and serum albumin (r=-0.41, p<0.05), oxLDL (r=-0.42, p<0.05) and overhydration (OH%, r=0.28, p<0.05). Moreover, administration of statins and/or angiotensin-converting-enzyme inhibitors was significantly related to a reduction of ADMA in KTRs. We have also identified a significant positive correlation between FGF-23 levels and inflammatory markers (hs-CRP, IL-6) and negative with overall index of renal function (eGFR-CKD EPI, eGFR-MDRD). Nutritional status, inflammation and endothelial dysfunction markers (ADMA, FGF-23) are considerably altered even in stable kidney transplant recipients.

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.

Dental caries and associated salivary biomarkers in patients with cystic fibrosis.

To evaluate the caries status of the Cystic fibrosis (CF) children and adolescents with the comparation of some biochemical markers, secretory-immunoglobulin-A (sIgA), and antimicrobial peptides in the saliva. In this cross-sectional descriptive study, the approval Ethics Board was obtained. Unstimulated saliva samples were collected from CF and healthy control children (non-CF) patients. Both groups underwent the same dental and periodontal evaluation scheme of the assessment. Human beta defensin (HBD1), human alpha defensin (HNP-1), cathelicidin (LL-37), sIgA in saliva were evaluated by enzyme-linked immunoassay method. A general biochemical analysis was performed. Statistical analysis was performed by using Statistical Package for the Social Sciences Version 20.0 (SPSS Inc.). A total of 21 (9 male, 12 female) CF and 23 (11 male, 12 female) control patients were participated with the mean age of 10.17 ± 3.38 and 9.52 ± 2.15 years, respectively. In control children, DMFT/S (decayed-missing-filled-tooth/surface-in-permanent-dentition), dmft/s (decayed-missing-filled-tooth/surface-in-primary-dentition) values were higher; DT (decayed-tooth in permanent dentition), ft (filled-tooth in primary dentition) and plaque index values were statistically significantly higher (p = 0.042, p = 0.005, p = 0.038, respectively) than CF patients. Bicarbonate was higher in control group; sodium, chloride, and total protein were higher in CF group; magnesium, calcium and phosphate levels were similar in each group (p &gt; 0.05). Alpha and beta defensin-1 levels in control group was statistically significantly higher (p = 0.037 and p = 0.020, respectively), while LL37 and sIgA were not statistically significantly higher (p &gt; 0.05) than CF group. Children with CF had lower caries in permanent teeth, filling in primary teeth, and an altered salivary biomarker profile, especially in HNB1, HNP1. Therefore, it is important to conduct periodic oral-dental controls among CF patients during their childhood.

The Role of Klotho G395A Gene Polymorphism in Atherosclerotic Cardiovascular Disease and Mortality Risk Scores in Non-dialysis Chronic Kidney Disease

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Klotho expression was reduced in patients with CKD, leading to vascular calcification, endothelial dysfunction, and atherosclerosis. We investigated the role of the klotho G395A gene polymorphism and plasma klotho level in the ten-year risk of atherosclerotic cardiovascular disease (ASCVD) and CVD mortality in CKD patients.METHODS: We used the PCR-CTPP assay method to genotype klotho G395A single nucleotide polymorphism (SNP) in 72 non-dialysis CKD patients. The klotho level was determined using the enzyme-linked immunoassay (ELISA) method. Path analysis was used to determine the relationship between the klotho G395A SNP, plasma klotho level, ASCVD risk score, and CVD mortality risk score.RESULTS: Our results showed that the GA genotype had lower plasma klotho levels than the GG genotype (path coefficient=-0.185, p=0.000). There was a significant negative correlation between plasma klotho level and the ASCVD risk score (r=-0.243, p=0.040), but no significant correlation was found between plasma klotho level and the CVD mortality risk score (r=-0.145, p=0.225). Path analysis showed that plasma klotho level had a significant negative direct effect on ASCVD risk score (path coefficient=-0.272, p=0.000) and an indirect effect on CVD mortality risk score (path coefficient=0.187, p=0.005).CONCLUSION: Klotho G395A SNP might reduce lower plasma klotho levels, which increased ASCVD and CVD mortality risk scores in non-dialysis CKD patients. However, other risk factors such as age, CKD stages, hypertension, and smoking should be taken into consideration. Therefore, large-scale genetic association studies with adjusted variables could be conducted in various ethnic groups for a more robust result.KEYWORDS: klotho, single nucleotide polymorphism, cardiovascular disease, chronic kidney disease

Development and validation of an isoform-independent monoclonal antibody–based ELISA for measurement of lipoprotein(a)

The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV2 and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV9. Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27–1,402 nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, 4076LETPTVV4082, on KIV9. In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.

8-Hydroxydeoxyguanosine Urine and Total Nitric Oxide Serum in Chronic Kidney Disease

Highlights: Oxidative markers 8-Hydroxydeoxyguanosine and Nitric Oxide was found high in hemodialysis and non-hemodialysis chronic kidney disease patients. There is no correlation between 8-Hydroxydeoxyguanosine and Nitric Oxide in hemodialysis and non-hemodialysis chronic kidney disease patients. Abstract: Oxidative stress is essential to chronic kidney disease (CKD). Several markers include 8-Hydroxydeoxyguanosine (8-OHdG) and Nitric Oxide (NO). Reactive oxygen species (ROS) and Reactive Nitrogen Species (RNS) increased in CKD and had a role in renal impairment progressivity. There are some controversies regarding oxidative markers in CKD patients in several studies. This study aimed to understand oxidative markers 8-OHdG and NO and explained the correlation of both markers in hemodialysis and non-hemodialysis CKD patients. Twenty hemodialysis patients and forty-nine non-hemodialysis patients were enrolled in this cross-sectional study. Urine patients were collected to measure 8-OHdG using the enzyme-linked immunoassay (ELISA) method, and NO was measured from serum patients using the Griss Saltzman method. Based on Bivariate Pearson analysis, there was no significant correlation between 8-OHdG urine and total NO serum in the hemodialysis group (p= 0,510, p&gt;0.05) and in the non-hemodialysis group (p= 0.801, p&gt;0,05). In this study, DNA oxidative marker, 8-OHdG, was not correlated with NO in CKD patients.

Homocysteine Levels and Osteoporotic Fracture in a Population Aged 55 Years Over in Medan District Indonesia

BACKGROUND: Osteoporosis is a chronic, progressive, systemic, skeletal, multifactorial disorder characterized by low bone mass, microarchitectural deterioration of bone tissues that increased the risk of fragility fracture. The increased prevalence of early osteoporosis in homocystinuria illustrates that homocysteine metabolism is involved in the process of osteoporosis. AIM: This study was aimed to look for the relationship between homocysteine levels in the blood and the occurrence of osteoporotic fractures in the population above 55 years old. METHODS: This is a descriptive study with cross-sectional approach, total sixty-five patients aged 55 years or older with osteoporotic fracture and as a control group without osteoporotic fracture. Blood pressure, heart rate, and body weight were recorded, bone density was measured using central dual-energy x-ray absorptiometry (DXA) machine/DXA and homocysteine plasma levels were measured using the enzyme-linked immunoassay method. RESULTS: Total of 65 samples with age range between 55 and 88 years, they were 38 peoples with osteoporosis, which 12 got the osteoporotic fracture. Among the 16 samples in osteopenia group, two got the fracture, among 11 samples with normal bone density, one got the osteoporotic fracture. There was statistical significant between fracture and bone mineral density (p = 0.00) and no statistical significant between bone density and gender (p = 0.08), bone density and body mass index (BMI) (p = 0.11). There was statistical significant correlation between homocysteine and age (p = 0.02), age and bone density (p = 0.002) but no statistical significant correlation between homocysteine and BMI (p = 0.07), homocysteine and osteoporotic fracture (p = 0.87). CONCLUSIONS: Homocysteine level did not increased the incidence of osteoporotic fracture, however homocysteine increased with aging and correlated with bone mineral density.

Development of Generic Immuno-Magnetic Bead-Based Enzyme-Linked Immunoassay for Ustiloxins in Rice Coupled with Enrichment.

Ustiloxins are a group of mycotoxins produced by rice false smut pathogen. Previous studies have shown that the false smut balls contain six types of ustiloxins, and these toxins are toxic to living organisms. Thus, immunoassay for on-site monitoring of ustiloxins in rice is urgently required. The current immunoassays are only for detecting single ustiloxin, and they cannot meet the demand for synchronous and rapid detection of the group toxins. Therefore, this study designed and synthesized a generic antigen with ustiloxin G as material based on the common structure of the mycotoxins. Ustiloxin G was conjugated to two carrier proteins including bovine serum albumin (BSA) and ovalbvmin (OVA) by carbon diimide method. The mice were immunized with ustiloxin-G-BSA to generate the antibody serum, which was further purified to obtain the generic antibody against ustiloxins. The conjugated ustiloxin G-OVA and generic antibodies were used for establishing the enzyme-linked immunosorbent assay (ELISA) for ustiloxin detection and optimizing experiment conditions. The characterization of the antibody showed that the semi-inhibitory concentrations (IC50) of ustiloxin A, B, and G were 0.53, 0.34, and 0.06 µg/mL, respectively, and that their corresponding cross-reactivities were 11.9%, 18.4%, and 100%, respectively. To increase ELISA detection efficiency, generic antibody was combined with magnetic beads to obtain sensitive and class-specific immune-magnetic beads. Based on these immuno-magnetic beads, a high-efficiency enzyme-linked immunoassay method was developed for ustiloxin detection, whose sensitivity to ustiloxin A, B, and G was improved to 0.15 µg/mL, 0.14 µg/mL, and 0.04 µg/mL, respectively. The method accuracy was evaluated by spiking ustiloxin G as standard, and the spiked samples were tested by the immune-magnetic bead-based ELISA. The result showed the ustiloxin G recoveries ranged from 101.9% to 116.4% and were accepted by a standard HPLC method, indicating that our developed method would be promising for on-site monitoring of ustiloxins in rice.