Enzyme-linked Immunosorbent Assay Method Research Articles

Comparison of RT.PCR and ELISA Methods in the Diagnosis of Human Cytomegalovirus in Kidney Transplant Patients

Background: In kidney transplant recipients prone to infections like cytomegalovirus (CMV), a vital need for an accurate diagnostic method is evident. This study at Khorshid Laboratory in Tehran rigorously compares real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) for CMV detection in transplant patients. Methods: In January to March 1400, 70 kidney transplant recipients were assessed for CMV DNA using RT-PCR and concurrent ELISA tests, with statistical analysis aided by SPSS. Results: In kidney transplant patients (average age: 49.40 ± 13.64 years), 4.3% tested positive for CMV via PCR. Strong correlation between serological and molecular methods. IgG and IgM antibody detection both showed high sensitivity and specificity, advocating for efficient CMV diagnosis at Khorshid Laboratory, Tehran. Conclusion: This study emphasizes the need for a quick and efficient CMV diagnostic approach in kidney transplant patients. The strong correlation between molecular and serological methods favors using the faster RT-PCR method, crucial for timely management, especially with the increasing age- related CMV incidence. The findings strongly recommend RT-PCR integration for enhanced sensitivity in kidney transplant patients at Khorshid Laboratory, Tehran.

Use of multivariant enzyme-linked immunosorbent assay (ELISA) in the diagnosis of autoimmune bullous disorders in a resource-limited setting: A single-center experience

Background Autoimmune blistering disorders (AIBD) result from the formation of auto-antibodies against adhesion proteins of the skin/mucosa(e). These auto-antibodies can be detected in the bound form in the tissue using direct immunofluorescence (DIF) or blood circulation using enzyme-linked immunosorbent assay (ELISA) or other methods. Objectives The objective of this study was to evaluate the concordance rate between the results of multivariant ELISA and the diagnosis of AIBD made using DIF and histopathology in an appropriate clinical context. Methods This was a retrospective study (December 2020 to April 2023) in which the multivariant ELISA assay (able to detect antibodies against desmoglein 1, desmoglein 3, BP180, BP230, envoplakin, and collagen VII) data were retrieved from the dermatology laboratory. Corresponding clinical and histopathology data were searched from relevant institutional databases. As per routine practice, the final diagnosis was assigned based on the clinical presentation, histopathology features and corresponding DIF report. Results After screening the records of 338 patients during the study period, 253 patients were included. Of them, 194 had AIBD and 59 had non-AIBD. In the autoimmune blistering disorder group, 122 and 72 patients had pemphigus and pemphigoid, respectively. Overall, a good level of agreement was found between multivariant ELISA results and the final diagnosis (Fleiss kappa = 0.631, p-value < 0.001). The pemphigus vulgaris group exhibited good agreement (kappa = 0.796, p < 0.001), while pemphigus foliaceous, bullous pemphigoid and non-autoimmune blistering disorders demonstrated moderate agreement (kappa = 0.641, 0.651, 0.533, respectively; p < 0.001). The mucous membrane pemphigoid group had a fair agreement (kappa = 0.289; p < 0.001). Limitations The limitations for the study were its retrospective design, fewer number of patients in certain groups like paraneoplastic pemphigus and gold-standard single antigen specific ELISA was not done. Conclusion Considering good agreement between the multivariant ELISA and the gold-standard diagnosis (clinical findings plus histopathology plus DIF), multivariant ELISA can be used for the diagnosis of AIBDs in places where facilities for DIF are unavailable. Multivariant ELISA can improve etiological diagnosis for a set of autoimmune blistering disorders whose target antigens are represented in the multivariant panel.

The fabrication of phosphotungstate@UIO-Au/reduced graphene oxidation for electrochemical ultrasensitive detection of alpha-fetoprotein.

As an early multi-purpose tumor marker of hepatocellular carcinoma, alpha-fetoprotein (AFP) plays a vital role in early diagnosis and treatment. To achieve the early and accurate determination of AFP, the POMOF was fabricated by embedding H3PW12O40 (PW12) into UIO-66-NH2, further immobilized on reduced GO (rGO) and fabricated an innovative POMOF nanocomposite (PW@UIO-Au/rGO) as an electrochemical immunosensor (ECI-sensor). The PW@UIO-Au/rGO achieved 17-fold signal enhancement owing to their synergistic effect, enabling PW@UIO-Au/rGO exhibit high oxidase-like catalytic activity, facilitating their sensing performance. Under optimal experimental conditions, the proposed PW@UIO-Au/rGO ECI-sensor presented excellent sensing performance over a wide range from 0.01 ng mL-1 to 500 ng mL-1 with ultra-low detection of 4.0 pg mL-1. Notably, sensing results in real serum samples were verified by the clinical enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence immunoassay (ECL) methods with excellent accuracy and consistency, indicating the excellent environmental tolerance of the proposed ECI-sensor. This work provided a promising strategy for designing feasible ultra-sensitive probe for sensing AFP in clinical test.

Abstract 4134273: Low 5-hydroxytryptamine levels are associated with adverse outcomes in patients with heart failure with preserved ejection fraction

Background: Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome and its pathophysiology is not fully understood. A monoamine, 5-hydroxytryptamine (5-HT), is involved in diverse biological functions and suggested to play a role in cardiovascular diseases. However, the clinical relevance of 5-HT in HFpEF remains unclear. Aims: This study aimed to elucidate the clinical significance and prognostic value of 5- HT in patients with HFpEF. Methods: We conducted a prospective study involving 240 consecutive hospitalized patients with HFpEF (mean age 72 years, 52% male). We measured circulating blood 5-HT levels using the enzyme-linked immunosorbent assay method. Clinical and outcome data were collected. Results: Correlation analysis revealed that 5-HT levels were negatively correlated with blood B-type natriuretic peptide concentration and tricuspid regurgitation pressure gradient. When patients were stratified into two groups based on the median 5-HT levels (90.5 ng/mL), Kaplan-Meier analysis showed that HFpEF patients with low blood 5-HT levels had lower event-free survival rates from the composite event of cardiac death and worsening heart failure over a median follow-up period of 725 days (Figure). In a multivariable Cox proportional hazard model adjusting for confounding variables, low levels of 5-HT were independently associated with increased risks of the composite of cardiac events (hazard ratio, 3.25; P < 0.01). Conclusion: Low 5-HT levels are associated with adverse outcomes in patients with HFpEF and 5-HT may serve as a useful biomarker for predicting prognosis in such patients.

Evaluation of protein impurities in Ademetionine 1,4-Butanedisulfonate

Ademetionine 1,4-Butanedisulfonate (SAMe) is widely used as a prescription drug to treat cholestasis associated with liver disease, and is also used to treat depression, Alzheimer's disease and other diseases. Currently, the main way to produce SAMe is by fermentation of S-adenosylmethionine synthetase（SAMS）. However, during the fermentation process, host cells produce contaminants unrelated to the treatment. Among them, host cell protein (HCP), which is co-purified with the drug, is the main impurity in the production process. HCP-induced antigenic reactions can lead to allergies or other adverse reactions and affect drug quality, efficacy, and safety. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) are currently used as the main analytical methods for measuring HCP. The ELISA method may ignore some HCPs with low immunogenicity, while mass spectrometry is primarily employed for the characterization of proteomes. Orthogonal methods can more effectively evaluate impurities in drugs. In this study, ELISA was initially utilized to quantify the content of Saccharomyces cerevisiae host protein in 11 batches of SAMe active pharmaceutical ingredient (API) from two suppliers, then the above APIs were qualitatively analyzed by MS method. Following the identification of surrogate peptide of SAMS, an Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry（UPLC-MS/MS）method was established and validated, the SAMS protein residues in 11 batches of samples were subsequently determined. The results demonstrated that the levels of Saccharomyces cerevisiae host protein and SAMS in the samples were both low, with values below 20 ppm and 1.19 ppm, respectively. The qualitative results also indicate that the types of peptide residues in the samples are more diverse than those of protein residues. Furthermore, residues of proteins and peptides can be detected in all samples, which underscores the importance of evaluating the HCP in API. This study employs a range of complementary detection methods to conduct a comprehensive and sensitive evaluation of total HCP and specific proteins in drugs, thereby guiding more informed assessments of the risks posed by HCP impurities in raw materials during pharmaceutical processes.

Digital microfluidic chip with photopatterned reactive sites for direct biomolecules immobilization and magnetic beads-free immunoassay

Digital microfluidics (DMF), as an emerging liquid-handling technology, is widely recognized as an ideal platform for enzyme-linked immunosorbent assay (ELISA). However, current DMF-based ELISA methods are highly dependent on magnetic beads (MBs), which usually involves tedious modification of MBs, complex peripherals for MBs control, and impairs the parallel processing capability of DMF. To address these issues, we herein developed a photopatterned reactive sites-integrated DMF chip (PRS-DMF) for MBs-free immunoassay. By taking advantage of click chemistry between N-(allyloxycarbonyloxy) succinimide and thiol-ene substrate, the preparation of PRS is very simple, and leaves the reactive N-hydroxysuccinimide (NHS) ester groups on the surface. This enables direct biomolecules immobilization without any surface modification/activation processes. Moreover, the patterned micropillar array can notably increase the specific surface area, and promote the liquid mixing and immunoreaction. As a consequence, highly sensitive and MBs-free ELISA can be readily achieved on PRS-DMF, with a proof-of-concept H5N1 assay having been successfully demonstrated herein, which offers a low limit of detection (LOD) of 147 pg/mL. This also avoids the use of complex magnetic peripherals, and thus, contributing to the instrument miniaturization into a very compact form. It is therefore reasonably expected that the proposed PRS-DMF may serve as a promising platform for a variety of biological and biomedical applications, especially in the field of point-of-care testing (POCT).

Application of Random Quality Control Based on Time -Series Model in ELISA Detection of Blood Donors in Nanjing

This study introduces a completely Random Quality Control (R-QC) method for Enzyme-Linked Immunosorbent Assay (ELISA) tests and compares it with traditional Fixed Quality Control (F-QC) methods across different testing items. A random quality control system was constructed using a Time-series model, enabling fully random positioning of quality control in ELISA tests. The Coefficient of Variation (CV) for R-QC were found to be lower as compared to F-QC in Hepatitis B surface antigen (HBsAg), anti-Human Immunodeficiency Virus (anti-HIV), and Treponema Pallidum (TP) items, and there were significant differences among them. Moreover, the alarm rates and out of control rates in the R-QC groups were notably lower than those in the F-QC groups. The experimentally designed R-QC method demonstrates superior stability, reducing alarm and out of control rates, with has significant implications for improving ELISA testing at the Nanjing Blood Center.

Effects of cholecalciferol supplementation on depressive symptoms, C-peptide, serotonin, and neurotrophin-3 in type 2 diabetes mellitus: A double-blind, randomized, placebo-controlled trial

The coexistence of depression and type 2 diabetes mellitus (T2DM) can significantly worsen disease prognosis and lower quality of life. Emerging evidence suggests that vitamin D deficiency contributes to the progression of T2DM and is closely associated with the development of depression. The aim of this study was to investigate the effects of cholecalciferol on depression in patients with T2DM, exploring its mechanisms by analyzing its impact on C-peptide, serotonin, and neurotrophin-3 levels. A double-blind, randomized, placebo-controlled clinical trial was conducted at Cipto Mangunkusumo General Hospital, Jakarta, Indonesia, from April 2021 to September 2022. Patients with T2DM and depressive symptoms were randomly assigned to two groups: received 4000 IU of cholecalciferol daily and received a placebo for 12 weeks. Depression was assessed using the Beck Depression Inventory-II (BDI-II) before and 12 weeks after the intervention. The levels of C-peptide, serotonin, and neurotrophin-3 were measured at the end of the fourth week of intervention using the enzyme-linked immunosorbent assay (ELISA) method. Between-group comparisons were made using independent Student t-tests and Mann-Whitney U tests. Paired Student t-tests or Wilcoxon tests were applied for within-group comparisons between pre- and post-intervention. A total of 70 T2DM patients with depression were included in this study, comprising 38 patients in the cholecalciferol group and 32 in the placebo group. C-peptide levels increased significantly in the cholecalciferol group compared to the placebo group (p=0.006). No significant differences were observed in serotonin and NT-3 levels between the cholecalciferol group compared to the placebo group. The cholecalciferol group had a significantly greater reduction in BDI-II scores compared to the placebo group (p<0.001). This trial highlights that taking cholecalciferol might help ease mild to moderate depression symptoms in patients with T2DM by enhancing c-peptide levels, though its effects on serotonin and neurotrophin-3 are still unclear.

The Effects of Traditional Asian Diet on Metabolism, Gut Microbiota, and Liver Tissue in NASH Rats

Traditional Asian Diets (AD) in rural areas have a significant risk of mortality due to cirrhosis and hepatocellular carcinoma as nonalcoholic fatty liver disease (NAFLD). This study aims to determine the relationship between AD and liver cancer cases using rat experimental animals Rattus norvegicus strain Wistar. The measured variables include metabolic parameters, gut microbiota profile, and liver histology. This study used 14 rats in two groups: Chow Diet (7 rats to CD) and AD (7 rats to AD), and were given the respective diets for 12 weeks. Enzyme-linked immunosorbent assay (ELISA) methods are used to analyze liver enzymes, lipid profiles, and blood sugar levels. The analysis of gut microbiota used variable region-specific 16S rRNA gene and V3-V4. Biopsy stained with Hematoxylin Eosin was used to study the histology of the liver. Moreover, it was analyzed utilizing NAS (NAFLD Activity Score). The result of this study indicated that reduce body weight the rats treated with AD significant different than treated with CD. Firmicutes, Lactobacillus reuteri, Prevotellaceae bacterium, Romboutsia ilealis, and Bacteroidota in AD greater than CD. Alzheimer's disease had notably higher levels of alkaline phosphatase compared to those diagnosed with Crohn's disease on individual diagnosis. Differences in total bilirubin, alanine transaminase, aspartate transaminase, blood sugar, total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides were not significant. The NAS analysis indicated that the two groups comprised rats lacking non-alcoholic fatty liver disease. Despite the high caloric content of the Asian diet, it did not lead to significant changes in metabolic parameters and liver histology related to non-alcoholic steatohepatitis. This behavior can be ascribed to the advantageous influence of the gut microbiota.

The expression of sICAM-1 influenced by Clonorchis sinensis co-infection in CHB patients.

Soluble Intercellular Adhesion Molecule-1 (sICAM-1) has emerged as an inflammatory biomarker of many essential functions. We investigated the level of sICAM-1 influenced by Clonorchis sinensis (C. sinensis) co-infection in chronic hepatitis B (CHB) patients to explore the degree of liver tissue inflammation and liver function damage after co-infection. The study included data from patients with C. sinensis mono-infection (n=27), hepatitis B virus (HBV) mono-infection (n=32), C. sinensis and HBV co-infection (n=24), post-hepatitis B liver cirrhosis (n=18), post-hepatitis B liver cirrhosis co-infected with C. sinensis (n=16), and healthy controls (n=39). The level of sICAM-1 was measured with specific enzyme-linked immunosorbent assay method. Compared to the healthy control group, all the experimental groups had significantly higher serum sICAM-1 levels. The levels of sICAM-1 in co-infected groups were significantly higher compared to the mono-infection groups and were positively correlated with the levels of glutamate aminotransferase (ALT) and aspartate aminotransferase (AST). Our research findings confirmed that co-infection could exacerbate liver tissue inflammation and liver function damage in patients, could raise the sICAM-1 level, and may lead to the chronicity of HBV infection. These results provide clues for pathological mechanism study and formulating treatment plans.

Correction of immune disorders under the influence of pharmacotherapy and extracorporeal magnetic stimulation with patients after surgical treatment of prostate adenoma

BACKGROUND: The course of benign prostatic hyperplasia is associated with immunological disorders at the systemic and local level. Despite the wide arsenal of pharmacological agents for the treatment of benign prostatic hyperplasia, some patients undergo surgical treatment. Postoperative management programmes should be aimed at reducing the risks of complications, including infectious-inflammatory complications. In this regard, the inclusion of immunomodulatory pharmacotherapy and therapeutic physical factors in the course of reconstructive treatment is reasonable and justified. AIM: To study the role of the complex effect of pharmacotherapy and extracorporeal magnetic stimulation in the correction of immune disorders with the patients after surgical treatment of benign prostatic hyperplasia. MATERIALS AND METHODS: There have been observed 88 patients after surgical treatment of benign prostatic hyperplasia, who underwent a medical rehabilitation at stages II and III. At stage II 43 patients in the comparison group underwent standardized drug therapy, individual exercise therapy; in the main group 45 patients additionally were prescribed immunomodulatory therapy with Tameritis. At stage III the patients of the comparison group continued pharmacotherapy, exercise therapy, and transrectal magnetic therapy; in the main group 45 patients were additionally prescribed extracorporeal magnetic stimulation. A method of enzyme-linked immunosorbent assay was used to study the content of interleukins (IL); IPSS-QOL (International Prostate Symptom Score – Quality-of-Life) and SF-36 (The Short Form-36) were used to assess the quality of life. Confidence level (р) 0.05. RESULTS: After the second inpatient stage of rehabilitation the patients of the main group after surgical treatment of benign prostatic hyperplasia had a decrease the level of pro-inflammatory IL by 47.5% on average (p 0.01) with an increase in the level of anti-inflammatory IL-10 by 36.2% (p 0.01). After the III outpatient rehabilitation stage, a positive dynamics significantly increased and amounted to 63.2% (p 0.01) and 51.9% (p 0.01), reaching the normative values in the population. It should be noted that the achieved results have been preserved in a long time. In the comparison group the positive dynamics was significantly lower (p 0.05; p 0.01) in all the cases after the II and III stages of rehabilitation. The patients of the main group were observed a decrease in the severity of urinary disorders (p 0.01), restoration of quality of life (p 0.05) according to IPSS-QOL and SF-36 questionnaires at significant inter-group values (p 0.05) with the same confidence. CONCLUSION: The integrated use of pharmacotherapy and extracorporeal magnetic stimulation with the patients after surgical treatment of benign prostatic hyperplasia provides significant correction of immune disorders, restoration of physical and mental health.

Unveiling connections: bisphenol A and vitamin D dynamics in breast milk among healthy lactating mothers

ABSTRACT Bisphenol A (BPA) has negative impact on human health. This study aims to explore the association between the levels of BPA and 25(OH) vitamin D in breastmilk. BPA exposure and vitamin D sources were recorded on the study form. Breastmilk 25(OH) vitamin D was measured by the competitive enzyme-linked immunosorbent assay method, and BPA was analyzed by fluorescence detector high pressure liquid chromatography technique. Multiple logistic regression analysis were performed for association. Among 70 lactating mothers, the median 25(OH) vitamin D level was 12.6 ng/mL. In 92.9% of breastmilk samples, detectable levels of BPA were found, with a median BPA concentration of 2.56 ng/mL. However, both univariate and multivariate analyses failed to reveal any association between breastmilk BPA and 25(OH) vitamin D levels. This lack of association in healthy lactating mothers, highlighting the need for further research to comprehensively understand the intricate dynamics of these factors.

Protective Effect of Lavandula officinalis on Pentylenetetrazol-Induced Epilepsy in Mice: Expression of NOS Genes and Caspase-3

Abstract Objective Given the incidence of epilepsy and the adverse effects of unconventional antiepileptic medicines, there is a need for a novel medical treatment strategy for epileptic patients. Material and Methods The current study involved the selection of 80 male mice, which were then separated into 10 experimental groups: pentylenetetrazol (PTZ), negative control which received normal saline, treatment which received Lavandula officinalis in two doses of 200 and 400 mg/kg, L. officinalis 200 mg/kg and 1400 w, L. officinalis 200 mg/kg and 7-NI, L. officinalis 200 mg/kg and diphenylene iodonium chloride (DPI), L. officinalis 400 mg/kg and 1400 w, L. officinalis 400 mg/kg and 7-nitroindazole (7-NI) and group which received L. officinalis 400 mg/kg and DPI. Each group was stimulated with an 11-day injection cycle (every 48 hours) of PTZ at a dosage of 35 mg/kg. All groups underwent PTZ challenge dosage (75 mg/kg) testing during the 12th injection. Ultimately, the brains of all mice were extracted, and the activity of genes related to neuronal nitric oxide, inducible nitric oxide, and endothelial nitric oxide was assessed. The enzyme-linked immunosorbent assay (ELISA) method was used to assess the quantity of caspase-3 in the groups. Results Lavandula officinalis decreased the severity of seizures. The findings of our study demonstrated that the extract had a suppressive effect on the expression of inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS; p < 0.05), while it had a stimulatory effect on endothelial NOS (eNOS; p < 0.05). In addition, L. officinalis reduced caspase-3 levels in the groups who were administered the extract. Conclusion The hydroalcoholic extract of L. officinalis has been found to be effective. Lavandula officinalis enhanced the expression of endothelial nitric oxide and reduced the levels of neuronal and inducible nitric oxide to a greater extent in brain tissue affected by epilepsy. The groups receiving extract derived from L. officinalis exhibited a reduction in the level of caspase-3.

Levels of three natural milk antimicrobial proteins during the manufacture of Cheddar cheese

Variations in cheese quality are explained by many factors. Among them, some influence the microbiota of cheeses such as native milk antimicrobial proteins. This project aimed to determine the concentration and activity of lactoferrin (LF), lactoperoxidase (LP) and lysozyme (LZ) during cheesemaking. Each antimicrobial protein was analyzed by two methods. An enzyme-linked immunosorbent assay (ELISA) technique was utilized to analyze each antimicrobial protein. In addition, LP was measured by spectrometry and LZ by fluorescence. Despite the lack of correlation between the results obtained by ELISA and the other methods, the measurement of LF, LP and LZ in raw milk and thermized milk showed no significant differences. The ELISA methods were selected to determine the retention of LF, LP and LZ in fresh cheese made from thermized milk. A total of 12 independent assays of cheddar cheese made commercially by a Canadian Dairy processor and at a pilot plant were evaluated. The concentration and activity of the three antimicrobial proteins were higher in cheese than in milk by an average factor of 3.5. These results allow a better understanding of the transition of the antimicrobial proteins present in milk when making cheese and to potentially, be able to control their concentration.

B-292 Detection of natural toxins derived from potatoes by biotin-streptavidin direct ELISA

Abstract Background α-solanine (SO) and α-chaconine (CHA) are natural toxins of potato and often cause food poisoning worldwide. We have previously developed a polyclonal antibody (anti-Sold antibody) that bind to SO and CHA and used it in an enzyme-linked immunosorbent assay (ELISA). Our previously constructed ELISA (conventional ELISA) has a weak point of low sensitivity in detecting SO and CHA, we have tried to improve it in this study. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Anti-Sold antibody was then purified from rabbit serum using affinity column chromatography. In our ELISA method, named “B-S direct ELISA”, biotin-labeled anti-Sold antibody is bound to SO and CHA in the sample, followed by the binding of peroxidase-labeled streptavidin. To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results The absorbance of the samples containing SO and CHA prepared in 10 mM phosphate buffer saline (PBS) was about 5-fold higher than that of the conventional ELISA. Furthermore, the absorbance of the samples containing SO and CHA prepared in human serum and urine was enhanced about 2.5- and 1.6-fold, respectively, compared to that of the conventional ELISA. The limit of detection in B-S direct ELISA was 0.32 ng/mL in 10 mM PBS, 2.08 ng/mL in serum, and 4.88 ng/mL in urine, respectively. Using this ELISA, it was also possible to measure the content of SO and CHA in extracts of potato tubers, peels, and sprouts. Both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.4- and 2.2-fold higher, respectively, in each part than those in the extracts from fresh potatoes. Furthermore, the potato sprout extract contained approximately 50-fold more SO and CHA than the tuber and 9-fold more than the peel. On the other hand, B-S direct ELISA cross-reacted with solanidine and solasodine, which have similar chemical structures to SO and CHA, and also with cholesterol and vitamin D3 in serum. Conclusions Based on our results, B-S direct ELISA presented greater performance in detecting SO and CHA than our conventional ELISA. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although this ELISA has a detection performance applicable to human biological samples, cross-reactivity should be fully considered when judging the results.

Can Productive Aptitude and Age Affect Circulating Serotonin, Total Thyroid Hormones, and Cortisol Patterns in Cows?

Cattle productivity, whether in terms of meat yield or milk production, is intricately regulated by a multitude of factors. Among them, hormone concentrations play a significant role, reflecting the complex interplay between endocrine regulation and physiological processes that ultimately determine the efficiency and yield of production. High concentrations of 5-hydroxytriptamine (5-HT) are associated with a reduced metabolic load at the onset of lactation and a lower milk yield. Thyroid hormones (THs) and cortisol also affect several metabolic pathways, including carbohydrate, protein, and lipid metabolism. The aim of this study was to assess if milk or meat aptitudes and age influence circulating 5-HT, THs, and cortisol concentrations, investigating the possible interactions among these parameters. The research was performed on 46 healthy cows of three different breeds. Enzyme-linked immunosorbent assay (ELISA) methods were used to quantify circulating 5-HT and cortisol concentrations, and an immunochemiluminescent analyzer was used for THs. For parameters exhibiting non-normal distributions, an ANCOVA model using age, aptitude, and their interaction as fixed factors was applied. Significant lower T3 concentrations were recorded in dairy cows than in meat cows. Moreover, T4 significantly decreased with advancing age both in cows aimed at milk and meat production. Lastly, T4 was positively correlated with T3 and 5-HT in meat production-oriented cows.

B-105 Farr Radioimmunoprecipitation Method Enhances Serial Monitoring of Quantitative Anti-dsDNA (double-stranded) Antibody Measurements in Lupus

Abstract Background Anti-double stranded DNA (anti-dsDNA) antibodies are an important criterion in the diagnosis, classification, and management of systemic lupus erythematous (SLE). High avidity anti-dsDNA IgG may be more clinically relevant and specific for SLE. As such, radioimmunoassay according to the Farr technique (Farr-RIPA) is often considered more sensitive and quantitative than enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae immunofluorescence assay (CLIFT) methods. Methods Measurements of anti-dsDNA antibodies in 147,016 serum patient samples were obtained by lab-developed Farr-RIPA. Serum with endogenous antibodies binds to 125I-radiolabelled dsDNA and undergoes subsequent immunoprecipitation under high salt conditions which selects for highly avidity anti-dsDNA antibodies. The analytic measurement range (AMR) of the assay is 5.7 to 85 IU/mL, and dilutional linearity is validated to 68,000 IU/mL for results >85 (ULOQ). Inter-assay precision is <10% CV. Results Of 147,016 measured samples, 5.4% (7800) were positive for anti-ds DNA antibodies (mean 251.7) with numeric values, ranging from 8.0 to 55,399 IU/mL. The numeric distribution of all positive results was such that 76% (5859/7800) were between 8.0 - 60 IU/mL, and the median was 19.4 with an interquartile range (IQR) of 36.1. A small percentage (3.9%) had very high anti-dsDNA levels greater than 1000 IU/mL and 0.4% (35/7800) were greater than 10,000 IU/mL. Serial measurements comprised almost half (45.5%) of all positives, occurring in 1016 unique patients an average of 3.5 levels within a 2-year time frame. A fifth of those patients, 200, were more frequently monitored with at least 4 levels. Of patients with the very highest dsDNA (>10,000), we analyzed the results of 17 unique patients who were frequently monitored an average of 6.1 times. Peak and nadir values yielded a mean delta of 20,751 with a mean of 264 days elapsing between peak and nadir. All except one showed a marked decrease in Anti-dsDNA over time with an average decrease of 15,685 IU/mL. Conclusions Here, we performed 147,016 Anti-dsDNA antibody measurements by Farr-RIPA, a precise method with a broad analytic range as compared to other methods which have a limited upper limit of quantitation or are semi-quantitative. Our 7800 positive Farr-RIPA results exhibited quantitative results from 8.0 to 55,399 IU/mL. The median of 19.4 and IQR of 36.1 showed most results to be <55.5 IU/mL. Very high levels >300 and >1000 IU/mL were seen in 10% and 4% of patient samples, respectively. Serial monitoring was frequent. In patients with the very highest levels >10,000, an-eight fold decrease in peak Anti-dsDNA levels was achieved over a mean of 264 days with more than half (9/17) achieving levels <250. The ability to detect and meaningfully monitor these patients is enhanced by the Farr-RIPA method. In conclusion, in 7800 positive samples, we demonstrated a wide range of inter- and intra-patient numeric Anti-dsDNA concentrations that denote the clinical usefulness of serial monitoring by Farr-RIPA.

Safety of raw milk in conditions of technogenic load

Abstract In the conditions of intensive development of the dairy industry, the issue of the content of potentially dangerous substances and inclusions in raw milk is acute. The paper presents data from an experiment to determine the content of microorganisms, mycotoxins, and toxic elements in raw milk. The research aims to monitor the safety indicators of milk obtained under conditions of technogenic load. Determination of microbiological indicators of milk safety was carried out using the classical microbiological method, the content of aflatoxin M1 - by the method of enzyme-linked immunosorbent assay [ELISA], the concentration of heavy metals was measured by the atomic absorption method, the presence of arsenic - by colorimetric method. As a result of the tests, it was found that raw milk obtained in the zone of significant environmental risks fully complies with the requirements of the technical regulations of the Customs Union.

Development of a multiplexing method for the quantification of “high-risk” host cell lipases in biotherapeutics by Luminex

Clearance of residual Host Cell Proteins (HCPs) is critical for the manufacturing processes of biotherapeutics. HCPs have the potential to impact product efficacy and quality, posing a risk to patient safety. It is therefore essential to be able to both identify and quantitate HCPs throughout drug development, even if the proteins are present in low concentrations. Traditional Enzyme-Linked Immunosorbent Assays (ELISAs) have historically served as the gold standard for monitoring HCPs; however, ELISA methods are labor-intensive and costly. With an increase of HCPs being identified below detectable quantification levels, there is a need for simultaneous detection of selectively targeted HCPs. Here, we develop a Luminex multiplexing method that is able to accurately quantify two “high-risk” lipases Lipoprotein Lipase (LPL) and Phospholipase B-Like 2 (PLBL2) within the same assay. This study outlines the method development for optimizing parameters such as antibody constructs, conjugation ratios, signal enhancement, and more in order to create the most efficient multiplexing method. As a result, a Luminex multiplexing method can provide a similar result to a monoplexing ELISA method but in a faster and more cost-effective manner. This method can be expanded to include other “high-risk” HCPs and used for future HCP applications.

Investigation of serum Metrnl levels in pregnant women with gestational diabetes mellitus: a prospective non-interventional cohort study.

The objective of this study was to investigate serum Metrnl levels in pregnant women with gestational diabetes mellitus and compare them with pregnant women without gestational diabetes mellitus. The gestational diabetes mellitus group consisted of 87 pregnant women diagnosed with gestational diabetes mellitus, and the control group consisted of 93 healthy pregnant women without gestational diabetes mellitus. Serum Metrnl levels were determined by the enzyme-linked immunosorbent assay method. The two groups were similar in terms of demographic features. The median serum Metrnl level was found to be 1.16 ng/mL in the gestational diabetes mellitus group, while it was determined as 2.2 ng/mL in the control group (p=0.001). The two groups were divided into two subgroups based on participants' body mass index, normal weight and overweight. The lowest median Metrnl level was detected in the normal weight gestational diabetes mellitus group, followed by the overweight gestational diabetes mellitus group, normal weight control group, and overweight control group (1.1, 1.2, 2, and 2.4 ng/mL, respectively). Receiver operating curve analysis was performed to determine the value of the serum Metrnl level in terms of predicting gestational diabetes mellitus. The area under the curve analysis of serum Metrnl for gestational diabetes mellitus estimation was 0.768 (p=0.000, 95%CI 0.698-0.839). The optimal cutoff value for serum Metrnl level was determined as 1.53 ng/mL with 69% sensitivity and 70% specificity. Serum Metrnl levels in pregnant women with gestational diabetes mellitus were found to be significantly lower than in pregnant women without gestational diabetes mellitus. The mechanisms underlying the decrease in serum Metrnl levels in gestational diabetes mellitus remain unclear for now, and future studies will reveal the role of Metrnl in the pathophysiology of gestational diabetes mellitus.