Competitive Chemiluminescent Immunoassay Research Articles

Vitamin B12 concentrations vary greatly in milk donated to a large provincial milk bank, and are influenced by supplementation and parity

Feeding parent's milk with supplemental donor milk (DM) is the optimal way to feed very low birth weight (VLBW) infants instead of formula; however, suboptimal neurodevelopment persists. This is believed due, in part, to suboptimal nutrition. Given vitamin B12's role in neurodevelopment and increased adoption of plant-based diets among females of child-bearing age, we aimed to determine the adequacy of vitamin B12 in DM (n=380 donors) and associated donor characteristics. Vitamin B12 was measured in consecutive raw DM donations received at the milk bank from March 2020-2021 using a validated competitive chemiluminescent enzyme immunoassay (IMMULITE 2000, Siemens). Donor characteristics were obtained from screening records and associations with milk vitamin B12 concentrations explored using a generalized additive model. Donors were 32±4 years old (mean±SD), and DM was expressed 98±85 days postpartum. Vitamin B12 concentrations in DM had a median (25th, 75th percentile) of 232 (149, 373) pmol/L; 64% had concentrations <310pmol/L (common cut-off for inadequacy in healthy term-born infants). In a multivariable analysis, donors consuming a vitamin B12-containing supplement had higher DM vitamin B12 (β±SE: 80.3±25.4pmol/L; p=0.020) compared to those not taking a supplement. Primiparous donors had higher DM vitamin B12 than multiparous donors (36.7±18.2pmol/L greater; p=0.044). No associations were observed for other donor characteristics. Milk donated to a large human milk bank showed evidence of suboptimal vitamin B12; levels were associated with both donor vitamin B12-containing supplement use and parity. Further research as to whether and when milk bank donors are recommended to consume a supplement and the benefits and risks of routine vitamin B12 supplementation of DM-fed infants is warranted.

Multivalent bifunctional nanobody to enhance the sensitivity of direct competitive chemiluminescence immunoassay for the detection of microcystin LR in lake water

Microcystin-LR (MC-LR), a toxic cyanobacterial toxin in freshwater, poses significant health and ecological risks due to its ability to induce cell apoptosis and liver damage. Sensitive detection of MC-LR is crucial for public health and water safety. In this work, we engineered a multivalent bifunctional nanobody (A2.3-C4-SBP) by fusing the anti-MC-LR nanobody gene (A2.3) with self-assembling peptides (C4) and a streptavidin-binding peptide (SBP). A2.3-C4-SBP was directionally immobilized on the ELISA microplate via streptavidin-mediated to develop a multivalent bifunctional nanobody-based chemiluminescent immunoassay (MBN-CLIA) for MC-LR detection in lake water. The IC50 of the A2.3-C4-SBP heptamer based CLIA was 5.80 ng/mL, and the LOD (IC10) was 0.33 ng/mL, which were 9.51-fold and 1.82-fold lower, respectively, than those of the A2.3-SBP monomer based CLIA. Additionally, the IC50 and LOD were 1.26-fold and 1.82-fold lower, respectively, than those of the A2.3-C4-SBP heptamer without streptavidin-mediated directional immobilization. In summary, this work developed a sensitive, rapid and simple immunoassay for the detection of MC-LR in lake water based on multivalent bifunctional nanobodies. Furthermore, the proposed combined strategy of nanobody multimerization and directed immobilization is simple to operate and has great potential to improve the sensitivity and signal amplification of various immunoassays.

The Prevalence of Vitamin D Deficiency and Associated Factors Among Subfertile Women: An Initial Experience From Northern Sri Lanka.

Subfertility is a global burden. Low vitamin D status has been associated with an increased risk for subfertility. Even though the prevalence of vitamin D deficiency is very high among women in Sri Lanka, not much importance is given to addressing the issue, especially among subfertile women. Moreover, no studies have been reported on the prevalence of vitamin D in subfertile women in Sri Lanka. Thus, the current study aimed to assess the prevalence and associated risk factors among subfertile women in the northern part of Sri Lanka. Methodology: This was a cross-sectional study comprising an interviewer-administered questionnaire and a laboratory analysis of vitamin D conducted on 371 subfertile women above 18 years of age who attended the subfertility clinic at the Professorial Unit in the Jaffna Teaching Hospital, Jaffna, Sri Lanka, between January 2023 to October 2023. Considering the time limitation and sample size, subfertile women attending during the study period were included without any sampling technique. Competitive chemiluminescence immunoassay for 25 hydroxy vitamin D (25(OH)D) was used to estimate the vitamin D level, and an external quality assessment (EQA) standard was used to categorize the level into deficient, insufficient, and sufficient. Chi-squared and ANOVA tests were used to assess the factors associated with vitamin D levels using IBM SPSS Statistics for Windows, Version 21.0 (Released 2012; IBM Corp., Armonk, New York, United States). Results: The mean age of subfertile women was 33.7 years (SD=6.7), and nearly half of them (n=173, 46.6%) were in the age group of 30-40 years. All the participants (n=371, 100%) were Sri Lankan Tamil, the majority (n=289, 77.9%) of them were Hindus, and nearly three-quarters of the participants (n=269, 72.5%) were unemployed. Moreover, almost 90% (n=334) of the subfertile women never estimated their vitamin D level prior to the study. The study revealed that only 36.4% (n=135) of subfertile women had adequate vitamin D levels, whereas 63.4% (n=235) had inadequate and low BMI levels, and intake of fish and eggs was statistically associated with adequate vitamin D levels. Conclusion: The study recommends a routine assessment of vitamin D levels, periodical monitoring of vitamin D levels for all subfertile women with particular attention towards the obese and overweight category, and a combination of pharmacological and dietary interventions to improve vitamin D levels, which might benefit the fertility outcome.

A-078 Harmonization of Immunoassay Reference Intervals with LC-MS/MS for Total and Calculated Free Testosterone Measurement

Abstract Background Management of male hypogonadism relies on accurate serum testosterone testing and reference values. This study aimed to establish harmonized reference ranges for total and calculated free testosterone in adults using the Siemens Atellica immunoassay, validated against a reference method. Methods The Siemens Atellica IM total testosterone assay is a fully automated competitive chemiluminescent immunoassay. We conducted 25 de-identified serum samples. Inter-laboratory correlation studies for total testosterone were performed on in-house Siemens Atellica and liquid chromatography-tandem mass spectrometry (LC-MS/MS) at a reference laboratory. Free testosterone was calculated using the International Society for the Study of the Aging Male (ISSAM) Free Testosterone calculator, which can be accessed from the ISSAM website, http://www.issam.ch/freetesto.htm, using the assayed concentrations of testosterone and Sex Hormone Binding Globulin (SHBG) on the Siemens Atellica IM system. The Siemens Atellica IM SHBG assay is a sandwich monoclonal immunoassay. Calculated free testosterone was compared with LC-MS/MS. Deming and linear regression, and Bland-Altman plot were employed for data analysis. Reference intervals were adopted from the reference laboratory and verified using 20 male and 20 female healthy samples. Total testosterone: Male 240-950 ng/dL, Female 8-60 ng/dL; Free testosterone: Male 2.29-20.7 ng/dL, Female 0-1.08 ng/dL. Results Strong correlations were observed between Siemens Atellica and LC-MS/MS for total testosterone (R^2 = 0.9915, slope = 0.988, intercept = 35) and for calculated free testosterone (R^2 = 0.9645, slope = 0.988, intercept = -0.4). Adopted reference intervals were verified using local healthy samples. Conclusions Harmonized reference intervals for total and calculated free testosterone, validated on Siemens Atellica, offer consistent results comparable to LC-MS/MS. This study demonstrates the feasibility of establishing standardized reference ranges for testosterone assays across laboratories.

B-291 Validation of a Simple Qualitative Immunoassay for 21 Drugs of Abuse in Meconium on a Single Biochip Array

Abstract Background Drugs use during pregnancy is a major social and medical issue, which may lead to significant pre- and post-natal complications, such as preterm birth and newborn mortality. Exposed newborns may have long-term behavioral, cognitive, and developmental problems. Accurate detection of in utero drug exposure is crucial for timely medical intervention for the newborn and the mother. Meconium is the ‘gold standard' matrix for detection of drugs in newborn which reflects drug exposure during pregnancy. This study aims to develop and optimize a clinical assay for simultaneous screening of 21 drugs in meconium using a custom biochip array. Methods Custom biochip array (Randox Laboratories Ltd.) included 21 discrete test regions (DTRs) for amphetamine, methamphetamine, opioids/opiates (targeting morphine, oxymorphone, oxycodone, hydrocodone, 6-monoacetyl morphine, tramadol, buprenorphine, fentanyl, and methadone), phencyclidine (PCP), cocaine (targeting benzoylecgonine), benzodiazepines (targeting oxazepam, lorazepam, and clonazepam), zolpidem, barbiturates (targeting phenobarbital), ethyl glucuronide, methylphenidate, and marijuana metabolite (targeting Delta-9-Carboxy-Tetrahydrocannabinol [THC-COOH]). Patient samples were obtained from the residual meconium samples that had been previously used for other tests. Blank meconium samples were spiked at in-house cutoff, negative and positive assay control levels. All samples were extracted in specimen diluent using Biotage® Lysera and centrifuged. The supernatant was analyzed on a fully automated Randox Evidence+® Analyzer. A robotic arm pipetted 60 µL sample on to a biochip and then the analyzer performed a competitive chemiluminescent immunoassay. The results were generated as relative light units (RLUs). Results The sample results were determined to be “positive” or “negative” based on average RLU of cutoff controls for each analyte on an assay. The intra-assay (CV ≤ 15%) and inter-assay (CV ≤ 18%) precision was 100% agreement for positive and negative controls and there was no observed carryover for any of the analytes. The sample recovery was excellent with minimal to no matrix suppression for all analytes. Accuracy was performed by comparing spiked analyte results from Randox Evidence+® Analyzer with the expected results and all the spiked samples matched (positive or negative). When authentic patient or spiked results were compared with external laboratory test results, some discordant results were obtained. However, the discrepancies could be explained based on the different cutoffs (e.g., the Randox assay had lower cutoffs) or the known assay cross reactivities within the analytes of same class. These deviations did not affect clinical utility of the assay. Conclusions We validated a novel and simple qualitative chemiluminescent immunoassay in meconium using a custom biochip array. Unlike other routine screening/ELISA methods, which require separate assays for detection of similar drug targets, the custom biochip array offers a convenient, rapid and efficient method that utilizes only a single sample preparation for the screening of all 21 drug targets. Therefore, this technology has potential to be utilized as high-throughput screening assay. Like all qualitative assays, the limitation of this test is that all positive results will need to be assessed quantitatively for a definitive interpretation.

Is There a Relationship of Cortisol and Sex Steroid Hormones With Mental Health in Middle-Aged Adults? The FIT-AGEING Study

ObjectiveMental health has emerged as a worldwide concern given the increasing incidence of anxiety and depression disorders in the last years. Cortisol and sex steroid hormones have been demonstrated to be important regulators of mental health processes in older adults. However, the evidence considering these integrated variables in apparently healthy middle-aged individuals has not been thoroughly addressed. The present study aimed to investigate the association of the plasma cortisol, testosterone, free testosterone, sex hormone–binding globulin (SHBG), and dehydroepiandrosterone sulfate (DHEAS) levels with mental health in middle-aged adults. MethodsThis cross-sectional study included a cohort of 73 middle-aged adults aged 45 to 65 years (women, 53%). Plasma cortisol, testosterone, SHBG, and DHEAS were assessed using a competitive chemiluminescence immunoassay. Free testosterone was calculated from the total testosterone and SHBG. Self-reported depression severity, generic health-related quality of life, hope, satisfaction with life, and optimism-pessimism were evaluated using the Beck Depression Inventory-II (BDI-II), 36-Item Short-Form Health Survey, Adult Hope Scale, Satisfaction with Life Scale, and Life Orientation Test-Revised, respectively—with higher total scores of these scales indicating greater levels of these variables. ResultsThe testosterone and free testosterone levels were inversely associated with the BDI-II values in men (all P ≤ .042). The cortisol levels were positively related with the Satisfaction with Life Scale scores, whereas the testosterone, free testosterone, SHBG, and DHEAS levels were negatively correlated with the BDI-II values in women (all P ≤ .045). ConclusionIn summary, these results suggest that the increased levels of steroid hormones—within the normal values—are associated with better mental health in middle-aged adults.

Vitamin D Deficiency Is Associated with Advanced Liver Fibrosis and Impaired Fasting Glucose in Alcohol Use Disorder.

Vitamin D deficiency is a risk factor for liver disease, insulin resistance, and beta cell dysfunction. Individuals with alcohol use disorder (AUD) have many comorbidities, with a heavy burden of liver disease and metabolic complications, including type 2 diabetes mellitus (T2DM). We aimed to analyze the prevalence and associations of vitamin D deficiency in patients admitted for in-hospital treatment of AUD. A cross-sectional study was conducted in patients consecutively admitted for the treatment of AUD between January 2017 and October 2023. Sociodemographic data, substance use characteristics, and blood parameters were available at admission. Vitamin D status was assessed through the serum concentrations of 25-hydroxyvitamin D [25(OH)D] levels using a direct competitive chemiluminescent immunoassay method. Deficiency of vitamin D was defined as a concentration less than 20 ng/mL; impaired fasting glucose (IFG) was defined by fasting blood glucose >100 mg/dL (5.6 mmol/L), and advanced liver fibrosis by an FIB-4 index >3.25. Two hundred and forty-three patients were included (75% male) with a mean age of 49 ± 10 years, mean BMI of 26.4 ± 7.3, mean alcohol consumption of 163 ± 81 g/day, and a mean duration of AUD of 18.1 ± 11.2 years. Mean 25(OH)D, fasting blood glucose, AST, ALT, and platelets were 14.4 ± 10.2 ng/mL, 103.4 ± 40.9 mg/dL, 55.1 ± 75.8 U/L, 44.8 ± 76.6 U/L, and 206.3 ± 84.8 × 109/L, respectively. The prevalence of vitamin D deficiency was 80.6%, and 41.1% of patients had levels less than 10 ng/mL. IFG was present in 32.3% of patients, and 20.5% had FIB-4 values >3.25. In the multivariable analysis, IFG (OR, 2.51; 95% CI: 1.02-6.17, p = 0.04) and advanced liver fibrosis (OR, 4.27; 95% CI: 1.21-15.0, p = 0.02) were the only factors associated with vitamin D deficiency. Vitamin D deficiency was very prevalent in this series of patients with AUD and was associated with impaired fasting glucose and advanced liver fibrosis.

Circulating 25-hydroxyvitamin D concentration can predict bowel resection risk among individuals with inflammatory bowel disease in a longitudinal cohort with 13 years of follow-up.

Although the beneficial properties of vitamin D in anti-inflammation and immunity-modulation are promising in the management of inflammatory bowel disease (IBD), data were limited for the critical IBD prognosis. The association between serum vitamin D levels and the risk of bowel resection in individuals with IBD remains largely unknown. We performed a longitudinal cohort study among 5474 individuals with IBD in the UK Biobank. Serum 25-hydroxyvitamin D [25(OH)D] was measured using direct competitive chemiluminescent immunoassay. Bowel resection events were ascertained via national inpatient data. Multivariable-adjusted Cox proportional hazard regression was used to examine the association between serum 25(OH)D and bowel resection risk, presented with hazard ratios (HRs) and 95% confidence intervals (CIs). Restricted cubic spline (RCS) was used to evaluate dose-response associations. During a mean follow-up of 13.1 years, we documented 513 incident bowel resection cases. Compared to participants with vitamin D deficiency, non-deficient participants showed a significantly reduced bowel resection risk in IBD (HR 0.72, 95% CI 0.59-0.87, P=0.001), Crohn's disease (CD, HR 0.74, 95% CI 0.56-0.98, P=0.038), and ulcerative colitis (UC, HR 0.73, 95% CI 0.57-0.95, P=0.020). When comparing extreme quintiles of 25(OH)D level, participants with IBD showed a 34% reduced risk of bowel resection (95% CI 11%-51%, P=0.007) and participants with UC showed a 46% reduced risk (95% CI 19%-64%, P=0.003), while this association was not significant in CD (HR 0.93, 95% CI 0.59-1.45, P=0.740). Linear dose-response associations were observed using the RCS curve (all P-nonlinearity>0.05). Increased serum level of 25(OH)D is independently associated with reduced bowel resection risk in IBD. This association was significant in UC but may not be stable in CD. Vitamin D deficiency is a risk factor for bowel resection in individuals with IBD, and may be an effective metric in predicting and risk-screening surgical events.

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

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

Low vitamin D levels are linked with increased cardiovascular disease risk in young adults: a sub-study and secondary analyses from the ACTIBATE randomized controlled trial.

Vitamin D deficiency is related to metabolic disturbances. Indeed, a poor vitamin D status has been usually detected in patients with cardiovascular disease (CVD). However, the relationship between vitamin D and CVD risk factors in young adults remains controversial at present. This study aimed to examine the association between circulating 25-hydroxivitamin D (25(OH)D) and CVD risk factors in young adults. The present cross-sectional study included a cohort of 177 young adults aged 18-25years old (65% women). 25(OH)D serum concentrations were assessed using a competitive chemiluminescence immunoassay. Fasting CVD risk factors (i.e., body composition, blood pressure, glucose metabolism, lipid profile, liver, and inflammatory markers) were determined by routine methods. A panel of 63 oxylipins and endocannabinoids (eCBs) was also analyzed by targeted metabolomics. Circulating 25(OH)D concentrations were inversely associated with a wide range of CVD risk factors including anthropometrical (all P ≤ 0.005), body composition (all P ≤ 0.038), glucose metabolism (all P ≤ 0.029), lipid profile (all P < 0.035), liver (all P ≤ 0.011), and pro-inflammatory biomarkers (all P ≤ 0.030). No associations of serum 25(OH)D concentrations were found with pro-inflammatory markers (all P ≥ 0.104), omega-6 and omega-3 oxylipins, nor eCBs concentrations or their analogs (all P ≥ 0.05). The present findings support the idea that 25(OH)D could be a useful predictor of CVD risk in young individuals.

Validation of a biochip array technology for multi-mycotoxins screening in Rice and other cereals

Cereal grains are widely cultivated agricultural commodities, and they constitute a key source of energy. However, many of them are affected by fungal contamination, and consequently, by the presence of mycotoxin. The development of monitoring techniques to evaluate exposure, is therefore of interest. Immunoassays have recently emerged as potential alternatives for the study of mycotoxins. This study focuses on a validation of a biochip array technology (BAT) for multi-mycotoxins screening in oat, barley, rye, and wheat grains. The Evidence Investigator Myco 7 (RANDOX Food Diagnostic), based in a competitive chemiluminescent immunoassay, was used for the simultaneous semi-quantitative detection of the mycotoxin's immunoassays. According to validation results, spiked rice samples showed low false results (5 % of false negatives and 5 % of false positives for fumonisins, 5 % of false negatives for Zearalenone, Aflatoxin B1, T2HT2 and Deoxynivalenol and 5 % of false positives were found for ochratoxin A). In the spiked samples of other cereals, were found 5 % of false positives for fumonisin B1+fumonisin B2, zearalenone, ochratoxin A and aflatoxin B1 and 5 % of false negatives were found for Fumonisin 1 + Fumonisin 2, Ochratoxin A, Aflatoxin B1 and T2+HT2 in agreement with European Union legislation performance criteria. This BAT immunoassay provides important benefits for the rapid and efficient screening of several mycotoxins in cereal grain samples at various levels.

Serum 25-hydroxyvitamin D, type 2 diabetes, and liver-related outcomes: Secondary data analysis of a prospective recruited cohort.

The association of vitamin D deficiency, which is prevalent in type 2 diabetes mellitus (T2DM), with liver disease and related mortality has not been quantified. Our study aimed to (1) investigate whether there is a synergistic association of vitamin D deficiency and T2DM with liver-related outcomes and (2) explore whether high 25-hydroxyvitamin D [25(OH)D] concentrations are associated with a lower risk of liver-related outcomes in T2DM. Leveraging the data from UK Biobank, we conducted 2 studies: study I assessed the joint associations of vitamin D deficiency [25(OH)D <50nmol/L] and T2DM with liver-related outcomes among 439,276 participants, and study II explored the associations of vitamin D status with liver-related outcomes among 21,519 individuals with T2DM. Baseline T2DM was identified through medication, laboratory test, and electronic health-related records. Serum 25(OH)D was measured by direct competitive chemiluminescent immunoassay. Liver-related outcomes included 6 liver disease end points and mortality by overall liver disease, chronic liver disease, and severe liver disease. During an average follow-up duration of 11.6 years, we observed a significant positive additive interaction effect (all synergy index>1.0) of T2DM and vitamin D deficiency on the risk of liver-related outcomes. Compared with participants without either T2DM or vitamin D deficiency, the multivariable-adjusted HRs of overall liver diseases were 1.29 for participants without T2DM but with vitamin D deficiency, 1.73 for participants with T2DM but without vitamin D deficiency, and 2.19 for participants with both T2DM and vitamin D deficiency. In individuals with T2DM, we observed that participants without vitamin D deficiency were inversely associated with incident liver disease and related mortality (multivariable-adjusted HRs 0.41-0.81) when compared with individuals with vitamin D deficiency. There are positive synergistic associations of vitamin D deficiency and T2DM with liver-related outcomes. Inverse associations between serum 25(OH)D concentrations and liver-related outcomes were observed in individuals with T2DM.

Effect of a subcutaneous implant of deslorelin acetate on serum testosterone concentrations in male Hermann's (Testudo hermanni sp.) and Greek (Testudo graeca sp.) tortoises.

Deslorelin acetate is a gonadotropin-releasing hormone agonist formulated in a controlled-release subcutaneous implant and designed for reversible suppression of testosterone production in dogs. It has also been demonstrated to be effective in other animal species, but no data on its effectiveness in male land tortoises are available. This study aimed to evaluate the effect of a 4.7-mg deslorelin acetate implant on serum testosterone concentrations in male Hermann's (Testudo hermanni sp.) and Greek (Testudo graeca sp.) tortoises. Twenty adult male tortoises housed under the same environmental conditions were enrolled for the study and randomly assigned to a treatment (D, n = 10) or a control (C, n = 10) group. Starting in May, males from the D group were implanted with a 4.7-mg deslorelin acetate device, whereas males from the C group did not receive any treatment. Blood samples were collected once immediately before implant application (S0-May) and at 15 days (S1-June), 2 (S2-July), and 5 (S3-October) months after application. Serum testosterone at each sampling time was measured through a solid-phase, enzyme-labeled, competitive chemiluminescent immunoassay. Median serum testosterone concentrations were not significantly different between the two groups in all sampling times, and no interaction between treatment and sampling time was observed. The present study, therefore, suggests that a single treatment with a 4.7-mg deslorelin acetate implant has no effect on testosterone circulation in male Hermann's and Greek tortoises during the following 5 months.

Association between 25(OH)D3 Levels and the Presence of COVID-19 Symptoms

Background: Recently, there have been studies reporting a relationship between vitamin D levels in the blood and the immune system. This study aimed to analyze the association between vitamin D levels and symptoms of coronavirus disease 2019 (COVID-19).Materials and methods: This study was an analytical survey study with a cross-sectional approach, with lecturers at Universitas Malahayati that have been infected with COVID-19 in 2022 as subjects. Total 47 subjects were included. Subjects were fasted overnight, then blood samples were taken from subjects in the next morning. The blood was centrifuged, then the serum was separated for examination by the direct competitive chemiluminescence immunoassay (CLIA) method using Architect 25-OH Vitamin D Reagent. Data was analyzed using logistic regression.Results: None of the subjects had normal blood levels of 25(OH)D3 and almost half (48.9%) of the subjects had symptoms when infected with COVID-19. There was a significant relationship between the level of 25(OH)D3 (p=0.001) and the status of the COVID-19 vaccine (p=0.013) with the presence of symptoms in COVID-19 patients.Conclusion: The lower the level of 25(OH)D3 in the blood and the more incomplete the COVID-19 vaccine, the greater the onset potential of COVID-19 symptoms. It is necessary to maintain vitamin D intake and increase the coverage of the COVID-19 vaccine, especially at booster doses.Keywords: vitamin D, COVID-19, vaccination, health protocol

Serum cotinine as a predictor of lipid-related indices in Turkish immigrants with type 2 diabetes: A clinic-based cross-sectional study.

Turkish immigrants form the largest ethnic minority group in the Netherlands and show a higher prevalence of (i) cardiovascular disease (CVD), (ii) cigarette smoking, and (iii) type 2 diabetes (T2D) as compared to the native Dutch. This study examines the association of CVD risk factors: serum cotinine, as an indicator of cigarette smoke, and lipid-related indices among first-generation (foreign-born) Turkish immigrants with T2D living in deprived neighbourhoods in the Netherlands. A total of 110 participants, physician-diagnosed with T2D, aged 30 years and older, were recruited by convenience sampling from the Schilderswijk neighbourhood of The Hague in a clinic-based cross-sectional design. Serum cotinine (independent variable) was measured with a solid-phase competitive chemiluminescent immunoassay. Serum lipids/lipoproteins (dependent variables) were determined by enzymatic assays and included: total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and triglycerides (TG). The Castelli Risk Index-I (CRI-I), and Atherogenic Coefficient (AC) were calculated using standardised formulas and assessed as dependent variables in multiple linear regression (MLR) models. Log-transformation of HDL-c, TG, CRI-I, and AC values were performed to account for the extreme right skewness of the data. Statistical analyses included descriptive characteristics and MLR models were adjusted for all major confounders of cotinine and lipids. The sample size had a mean age of 52.5 years [standard deviation (SD) = 9.21]. The geometric mean of serum cotinine level was 236.63 ng/mL [confidence interval (CI) = 175.89 ± 318.36]. The MLR models indicated that high serum cotinine levels (≥10 ng/mL) was positively associated with HDL-c (P = 0.04), CRI-I (P = 0.03), and AC (P = 0.03) in the age, gender, WC, diabetes medications, and statins-adjusted models (n = 32). This study indicated that lipid ratios of HDL-c, CRI-I and AC are dependent determinants of serum cotinine and higher serum cotinine levels (≥10 ng/mL) are associated with worse HDL-c, CRI-I and AC values in participants with T2D. Clinical comprehension of these biochemical indicators (lipids/lipoproteins) and symptomatic results (CVD risk) in individuals with T2D will aid in the intervention (smoking) approach for this vulnerable cohort (Turkish immigrants). Therapy that is targetted to modify this behavioural risk factor may improve cardiovascular health outcomes and prevent comorbidities in Turkish immigrants with T2D living in deprived neighbourhoods in the Netherlands. In the meantime, this report contributes to a growing body of information and provides essential guidance to researchers and clinicians.

Development of indirect competitive ELISA and CLEIA for quantitative analysis of melatonin in health products.

Melatonin (MT), as a hormone regulating the rhythm of sleep, is widely used in health products. However, illicit and excessive use of MT might cause undesirable effects. Therefore, it is essential to establish highly sensitive and specific rapid methods for MT analysis in health products. In this study, we established indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for sensitive and selective detection of MT in health products. Under optimal conditions, half-maximum inhibitory concentration (IC50 value) and limit of detection (LOD, IC10 value) for MT by ic-ELISA were 0.25 ng mL-1 and 0.03 ng mL-1 respectively, while the IC50 and LOD of ic-CLEIA were lower at 0.17 ng mL-1 and 0.03 ng mL-1 respectively. Three MT-free Chinese patent medicines were spiked with MT and the recovery rates ranged from 71.89% to 117% (ic-ELISA) and 83.66% to 107.17% (ic-CLEIA). The level of MT in six MT-containing health products was assessed in parallel using the developed methods and HPLC. Both ic-ELISA and ic-CLEIA showed good consistencies (R2 = 0.999 and 0.993, respectively) with HPLC, indicating that the two methods developed were sensitive, fast, and reliable for application in MT analysis.

Serial changes in the concentrations of cortisol and thyroid hormones in Beagle dogs infected with Babesia rossi

An experimental infection using Babesia (B.) rossi was performed in healthy male Beagle dogs to assess the changes in endocrine variables during disease. Two dogs were infected with a low dose (LD) of parasite inoculum (104 parasites) and three dogs were infected with a high dose (HD) (108 parasites). Basal serum cortisol, thyroxine (T4), and thyrotropin (TSH) concentrations were measured every second day. Samples were analyzed using a solid- phase, competitive chemiluminescent enzyme immunoassay (Immulyte® 2000, Siemens). Variables were compared between groups and timepoints using linear mixed models. In both groups, the median cortisol concentration increased, whilst the median T4 concentration decreased after infection, with a return towards baseline concentration post treatment. The highest cortisol and the lowest T4 concentrations were reached at 96 h and 108 h post infection, respectively, in the HD group and slightly later at 108 and 144 h post-infection, respectively, in the LD group. A higher cortisol concentration with a more rapid increase, and a lower T4 concentration with a more rapid decline, were associated with disease severity and a higher dose of parasite inoculum. The TSH concentration remained within the reference interval throughout the study period. This study illustrated the temporal changes in endocrine parameters during experimental B. rossi infection and demonstrated that cortisol and T4 tracked the severity of disease, albeit in opposite directions.

Automated detection of neutralizing SARS-CoV-2 antibodies in minutes using a competitive chemiluminescence immunoassay

The SARS-CoV-2 pandemic has shown the importance of rapid and comprehensive diagnostic tools. While there are numerous rapid antigen tests available, rapid serological assays for the detection of neutralizing antibodies are and will be needed to determine not only the amount of antibodies formed after infection or vaccination but also their neutralizing potential, preventing the cell entry of SARS-CoV-2. Current active-virus neutralization assays require biosafety level 3 facilities, while virus-free surrogate assays are more versatile in applications, but still take typically several hours until results are available. To overcome these disadvantages, we developed a competitive chemiluminescence immunoassay that enables the detection of neutralizing SARS-CoV-2 antibodies within 7 min. The neutralizing antibodies bind to the viral receptor binding domain (RBD) and inhibit the binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. This competitive binding inhibition test was characterized with a set of 80 samples, which could all be classified correctly. The assay results favorably compare to those obtained with a more time-intensive ELISA-based neutralization test and a commercial surrogate neutralization assay. Our test could further be used to detect individuals with a high total IgG antibody titer, but only a low neutralizing titer, as well as for monitoring neutralizing antibodies after vaccinations. This effective performance in SARS-CoV-2 seromonitoring delineates the potential for the test to be adapted to other diseases in the future.Graphical

Re-evaluation of the low-dose dexamethasone suppression test in dogs.

This study aims to re-evaluate the low-dose dexamethasone suppression test 8-hour cortisol cut-point for the diagnosis of hypercortisolism in dogs using a solid-phase, competitive chemiluminescent enzyme immunoassay. Twenty-seven client-owned dogs with naturally occurring hypercortisolism and 30 healthy control dogs were prospectively recruited. Performance of the low-dose dexamethasone suppression test was assessed using sensitivity, specificity and a receiver operating characteristic curve compared to a clinical diagnosis of hypercortisolism including response to treatment. Twenty-four dogs were diagnosed with pituitary-dependent hypercortisolism and three with adrenal-dependent hypercortisolism. In 30 healthy control dogs, 8-hour post-dexamethasone cortisol concentrations ranged from 5.5 to 39 nmol/L. A receiver operating characteristic curve curve constructed from the 8-hour post-dexamethasone cortisol concentrations of hypercortisolism and control dogs demonstrated that the most discriminatory cut-point was more than 39 nmol/L with sensitivity of 85.2% (95% confidence interval, 67.5% to 94.1%) and specificity of 100% (95% confidence interval, 88.7% to 100.0%) and an area under the curve of 0.963. The optimal cut-point of more than 36 nmol/L proposed by this study is similar to the currently accepted 8-hour cortisol concentration cut-point for diagnosing hypercortisolism when using a solid-phase, competitive chemiluminescent enzyme immunoassay.

Production of high affinity monoclonal antibody and development of indirect competitive chemiluminescence enzyme immunoassay for gentamicin residue in animal tissues

To determine gentamicin residues in animal tissues, a monoclonal antibody (Mab) was produced and a sensitive indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. At first, gentamicin was conjugated with bovine serum albumin as immunogens which were used to immunize BALB/c mice. Then, an anti-gentamicin Mab was prepared by hybridoma technology. Finally, a sensitive icCLEIA was developed with an 50% inhibition concentration (IC50) of 0.067 ng/mL for gentamicin. The limit of detection of the icCLEIA was 0.002 ng/mL. The cross reactivity of the Mab with structural analogues were<0.01%. The recoveries of gentamicin ranged from 80 to 101% and coefficient of variation was <6.4% in pork and fish samples. Samples were detected by UPLC-MS/MS for evaluating reliability of the icCLEIA. The results suggested that the prepared anti-gentamicin Mab can be used for rapid and convenient immunoassays to detect gentamicin residues in animal tissues.