Use Of Enzyme-linked Immunosorbent Assay Research Articles

274 Evaluation of recombinant Capripoxvirus antigens in the development of indirect enzyme linked immunosorbent assays

Abstract Capripoxvirus is a genus of DNA viruses that includes sheep pox virus, goat pox virus and lumpy skin disease virus (LSDV). These viruses are considered high consequence viruses due to the severe clinical disease leading to economic losses to production and impact on trade. The recent spread of LSDV in Europe and Asia has demonstrated the transboundary nature of LSDV. The vaccines used to control Capripoxvirus infections currently do not have the ability to differentiate infected from vaccinated animals (DIVA) and the enzyme linked immunosorbent assays (ELISA) available for detection of antibodies to Capripoxvirus infections are not ideal. Previous research has been done to evaluate different Capripoxvirus antigens using E. coli expressed antigens; however, there has been no prior evaluation of baculovirus expressed Capripoxvirus antigens for use in ELISA. The objective of the current research was to produce 6 recombinant Capripoxvirus antigens using a baculovirus expression vector system (BEVS) and to evaluate these antigens in ELISAs to test bovine, sheep, and goat serum. The sequences used to produce the recombinant antigens were based on a consensus obtained using reference genomes consisting of all three viruses within the genus. All six antigens were produced and purified in-house using BEVS followed by benchtop or FPLC purification (Figure 1). To date, the antigens have been fully validated for performance in indirect ELISA using sheep sera (Figure 2) and partially using cattle sera, with goat sera validation to follow. The ELISAs were developed and optimized using known reference sera as controls. The performance of the ELISAs developed was assessed in comparison with the only commercially available Capripoxvirus serological test. The results demonstrate that some of the antigens used for the ELISAs completely outperformed the commercial test. Several antigens were found to be useful for ELISAs to detect responses in sheep and cattle sera. The sensitivities and specificities of the different ELISAs for sheep sera ranged from 99.54% to 53.57% sensitivity and 99.54 to 94.95% specificity with likelihood ratios above 10 using receiver operating characteristic curve analysis, while the commercial kit had a 42.85% sensitivity using the same positive sheep serum panel evaluated. The development of these serological assays allows for the simultaneous evaluation of immunogenicity of Capripoxvirus antigens through the testing of serum from experimentally infected animals. This study demonstrates the proof of principle, that sensitive and specific ELISAs can be developed for Capripoxviruses.

Antiphospholipid Antibody Testing in a Maximum Care Hospital: Method-Dependent Differences.

Background: Antiphospholipid antibody (aPL) testing is critical for the classification of antiphospholipid syndrome. The 2023 ACR/EULAR classification criteria recommend the use of enzyme-linked immunosorbent assays (ELISAs) and specific thresholds for aPL positivity. Since non-ELISA methods are increasingly used, we compared and evaluated ELISA and non-ELISA aPL assays in a real-world maximum care hospital setting. Methods: Between January 2021 and June 2024, anticardiolipin (aCL; IgG and IgM) and anti-beta2 glycoprotein I (aß2GPI; IgG and IgM) antibodies were measured using ELISA (n = 5115) and a chemiluminescence-based automated immunoassay (CLIA) (n = 3820). Results of parallel testing were compared, and associations with clinical and laboratory characteristics were evaluated. Results: A total of 946 samples were tested using ELISA and CLIA in parallel. A total of 136 (14%) specimens were positive for at least one aPL, and 55 (6%) specimens were from patients diagnosed with APS. Among the latter, 47 (85%) and 41 (75%) patients were positive when ELISA- or CLIA-based aPL assays were used, respectively. After applying the >40 units threshold of the new classification criteria, the number of aPL-positive specimens was significantly lower. In the entire cohort, the agreement between ELISA and CLIA aPL assays was acceptable only for aß2GPI IgG; the results from the two methods did not agree for aCL IgG/IgM and aß2GPI IgM. In APS patients, the agreement between ELISA and CLIA aPL assays was acceptable for aß2GPI IgG and IgM but poor for aCL IgG and IgM. Antibody levels in APS patients were significantly higher using CLIA compared to ELISA. Conclusions: The method-dependent discrepancies between ELISA- and CLIA-based aPL assays regarding the quantitative and qualitative results are substantial. Both methods are suitable for APS classification, but the choice of aPL assay may influence the classification, and therefore, aPL results should be interpreted carefully in the clinical context.

Understanding the pharmacokinetic journey of Fc-fusion protein, rhIL-7-hyFc using complementary approach of two analytical methods, accelerator mass spectrometry and ELISA.

Antibody-based therapeutics (ABTs), including monoclonal/polyclonal antibodies and fragment crystallizable region (Fc)-fusion proteins, are increasingly used in disease treatment, driving the global market growth. Understanding the pharmacokinetic (PK) properties of ABTs is crucial for their clinical effectiveness. This study investigated the PK profile and tissue distribution of efineptakin alfa, a long-acting recombinant human interleukin-7 (rhIL-7-hyFc), using enzyme-linked immunosorbent assay (ELISA) and accelerator mass spectrometry (AMS). Totally, four rats were injected intramuscularly with 1mg/kg of rhIL-7-hyFc containing 14C-rhIL-7-hyFc, which was prepared via reductive methylation. Serum total radioactivity (TRA) and serum rhIL-7-hyFc concentrations were quantified using AMS and ELISA, respectively. The TRA concentrations in organs were determined by AMS. Serum TRA peaked at 10hours with a terminal half-life of 40hours. The rhIL-7-hyFc exhibited a mean peak concentration at around 17hours and a rapid elimination with a half-life of 12.3hours. Peak concentration and area under the curve of TRA were higher than those of rhIL-7-hyFc. Tissue distribution analysis showed an elevated TRA concentrations in lymph nodes, kidneys, and spleen, indicating rhIL-7-hyFc's affinity for these organs. The study also simulated the positions of 14C labeling in rhIL-7-hyFc, identifying specific residues in the fragment of rhIL-7 portion, and provided the explanation of distinct analytes targeted by each method. Combining ELISA and AMS provided advantages by offering sensitivity and specificity for quantification as well as enabling the identification of analyte forms. The integrated use of ELISA and AMS offers valuable insights for the development and optimization of ABT.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

Human neural stem cells repress glioma cell progression in a paracrine manner by downregulating the Wnt/β-catenin signalling pathway.

Neural stem cells (NSCs) play crucial roles in neurological disorders and tissue injury repair through exerting paracrine effects. However, the effects of NSC-derived factors on glioma progression remain unclear. This study aimed to evaluate the effects of human NSC-conditioned medium (NSC-CM) on the behaviour of glioma cells using an in vitro co-culture system. Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays revealed that NSC-CM inhibited glioma cell proliferation and growth in a foetal bovine serum (FBS)-independent manner. In addition, our wound healing assay demonstrated that NSC-CM repressed glioma cell migration, while results from transwell and 3D spheroid invasion assays indicated that NSC-CM also reduced the invasion capacity of glioma cells. Flow cytometry showed that NSC-CM prevented cell cycle progression from the G1 to S phase and promoted apoptosis. Western blotting was used to show that the expression of Wnt/β-catenin pathway-related proteins, including β-catenin, c-Myc, cyclin D1, CD44, and Met, was remarkably decreased in NSC-CM-treated glioma cells. Furthermore, the addition of a Wnt/β-catenin pathway activator, CHIR99021, significantly induced the expression of β-catenin and Met and increased the proliferative and invasive capabilities of control medium-treated glioma cells but not those of NSC-CM-treated glioma cells. The use of enzyme-linked immunosorbent assays (ELISA) revealed the secretion of some anti-tumour factors in human and rat NSCs, including interferon-α and dickkopf-1. Our data suggest that NSC-CM partially inhibits glioma cell progression by downregulating Wnt/β-catenin signalling. This study may serve as a basis for developing future anti-glioma therapies based on NSC derivatives.

Osteoprotegerin and Interleukin-37 are Correlated with Liver Diseases in Chronic Hepatitis B Virus (HBV)-infected Subjects

BACKGROUND: Various biological markers have been proposed to predict subclinical events in subjects with chronic hepatitis B virus (HBV). However, studies regarding chronic HBV infection are still limited. The current study aimed to investigate the relationship between osteoprotegerin (OPG) and interleukin-37 (IL-37) serum levels in chronic HBV subjects.METHODS: Sixty subjects with chronic HBV infection without previous treatment and 30 healthy subjects were included in this study. Blood samples were withdrawn and examined for biochemical parameters through the use of enzyme-linked immunosorbent assay (ELISA) assessments. Moreover, anthropometric estimations and medical histories were performed on all subjects through a standard self-administered questionnaire.RESULTS: A highly significant elevation (p<0.01) in serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and IL-37 and a significant increase (p<0.05) in serum level of OPG were observed in chronic HBV subjects compared with the controls. The data demonstrated that OPG had a positive correlation with IL-37 and that both OPG and IL-37 had significantly positive correlations with ALT and AST. Furthermore, the area under the receiver operating characteristics (ROC) curve (AUC) was computed for ALT, AST, OPG, and IL-37 (AUC = 0.831, 0.829, 0.608, and 0.618, respectively) which could be potentially greater predictive biomarkers in chronic HBV subjects.CONCLUSION: The positive correlation of OPG and IL-37 with ALT and AST and the high positive value of AUC for OPG and IL-37 shows that OPG and IL-37 could to be potential inflammatory biomarkers for the early onset of liver disease and hepatocellular carcinoma in chronic HBV-infected subjects.KEYWORDS: osteoprotegerin, interleukin-37, hepatitis B virus, inflammation, liver

Evaluation of glutamic acid decarboxylase (GAD) 65 antibody detection methods for neurological and diabetic investigation in an Australian diagnostic laboratory

The role of anti-glutamic acid decarboxylase (GAD) 65 autoantibodies in autoimmune neurological conditions is evolving, but testing recommendations remain unchanged in Australia with GAD enzyme-linked immunosorbent assay (ELISA) and immunoblot as the only two Therapeutic Goods Administration approved testing methods available in Australia. Common practice is for use of ELISA in diagnosis of type 1 diabetes mellitus (T1DM) and use of immunoblot for diagnosis of GAD65-associated neurological disease. We observed a cohort of patients with negative immunoblot results and positive ELISA in the context of GAD-associated neurological disease without T1DM. In the absence of robust consensus guidelines on preferred testing modalities, we sought to determine if ELISA could have a superior role in the diagnosis of GAD-associated neurological disease when compared to immunoblot in this paper. We tested for anti-GAD65 autoantibodies on 55 patient samples, 40 samples requested for neurological disease and 15 type 1 diabetes samples with detectable anti-GAD65, using two testing platforms: Euroimmun anti-GAD enzyme-linked immunosorbent assay (ELISA) and. Euroimmun EuroLine immunoblot for paraneoplastic neurologic syndromes. These results were correlated against the clinical scenario. Positive ELISA results had a sensitivity of 100% and specificity of 91% for GAD65-related neurological disease. Immunoblot showed sensitivity of 43% and specificity of 76% for GAD65-related neurological disease. ELISA proved more sensitive and specific for GAD65-related neurological disease compared to immunoblot, raising questions about the role of this testing modality in neurological disease. We propose that ELISA should be used as a sole diagnostic method for all GAD65 antibody-related neurological disease over immunoblot. The presence of anti-GAD65 antibody on immunoblot is of doubtful clinical significance.

Nesfatin-1 as a potential marker for functional hypothalamic amenorrhea

Objective Nesfatin-1 plays an important role in regulating metabolism, appetite, gut motility, and eating behavior. It is suspected that abnormalities in nesfatin-1 secretion may be involved in the development of anorexia nervosa, and as such, this study aims to investigate the “circumstances of” nesfatin-1 in patients with functional hypothalamic amenorrhea (FHA). Materials and Methods One hundred and forty-seven patients with FHA were enrolled to the present study. A control group consisting of 88 healthy, age-matched subjects was used. Both study and control groups had blood samples drawn to establish baseline serum concentrations of luteinizing hormone, follicle-stimulating hormone, estradiol, prolactin, thyroid-stimulating hormone, fT4, morning cortisol, dehydroepiandrosterone sulfate, testosterone, glucose, and insulin. Nesfatin-1 was also measured with the use of enzyme-linked immunosorbent assay. Results Patients with FHA were found to have a significantly decreased concentration of serum nesfatin-1 when compared to healthy controls (6.21 ± 4.79 vs. 8.64 ± 6.63 respectively, p = 0.005). No statistically significant difference in nesfatin-1 levels was found between patients with normal and decreased BMI in the FHA group. Significant positive correlation was observed between serum nesfatin-1 concentration and 17-β-estradiol, while a significant negative correlation was observed between serum nesfatin-1 concentration and patient age, fasting glucose, and HDL levels. Conclusions This is the first known study to examine nesfatin-1 concentration in the context of clinical FHA. Patients with FHA were found to have decreased serum nesfatin-1 concentrations. This finding may prove instrumental in our future approach managing patients with FHA.

Determination of Helicobacter Pylori Infection Prevalence by Non-invasive Methods

Background: Helicobacter pylori (H. pylori) is identified as the most frequent agent of bacterial infections in humans which can cause various gastrointestinal diseases. This pathogen has infected approximately half of the world’s population, and its outbreak has varied across different regions. The purpose of this study was to estimate the H. pylori infection prevalence amongst patients in Fardis county, Alborz province, Iran, using noninvasive methods. Methods: A total of 5677 patients were analyzed from September 2020 to October 2021 to detect H. pylori by the use of enzyme-linked immunosorbent assay (ELISA) IgG, IgA, and IgM tests, stool antigen test (SAT), and urea breath test (UBT). Results: Of 5677 patients, 3486 (61.4%) were female and 2191 (38.6%) were male with the mean age of 38.82 ± 18.289 years old. The overall rate of H. pylori infection positive was 31.46%, and the serological tests were the most prescribed types of tests. The IgG test and then SAT detected the most positive cases. Further, the infection rate was significantly associated with age. Except for the case of IgM, which was higher in females compared to males, no significant difference was found between gender and bacteria outbreak. Conclusions: This study indicated a decline in H. pylori infection prevalence compared with the prior survey conducted at this center. However, its rate amongst the patients referring to Fardis laboratory is still high.

Serum Calprotectin - a NET Product - as a Biomarker of Disease Activity in Patients with Systemic Lupus Erythematosus: A Single-Center Case-Control Study from Poland.

BackgroundCalprotectin (S100A8/A9 or myeloid-related protein 8/14) is a heterodimeric S100 complex expressed in leukocytes. Calprotectin participates in development of the inflammatory response by binding to receptors for advanced glycation end-products (RAGE) and Toll-like receptors (TLR). The clinical activity of systemic lupus erythematosus (SLE) is evaluated using the Systemic Lupus International Collaborating Clinics (SLICC) criteria and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). This Polish single-center case-control study aimed to evaluate serum levels of calprotectin as a rapid diagnostic biomarker of SLE (59 patients with SLE were compared with 52 healthy controls).Material/MethodsCalprotectin concentration was measured with the use of enzyme-linked immunosorbent assay (ELISA). The SLE activity of the patients was assessed by the SLEDAI scale. Statistical analysis of the results was carried out using MedCalc 15.8 software. P<0.05 was considered statistically significant.ResultsA significantly higher concentration of calprotectin was found in the study group compared to the control group (medians: 3.11 vs 2.45 ng/ml; P=0.0013). We found that calprotectin has high sensitivity (89.83%) and specificity (53.85%) in differentiating between SLE patients and healthy volunteers. We found that calprotectin has very high sensitivity (100%) and specificity (82.46%) in detection of patients with moderate and severe SLE assessed using SLEDAI.ConclusionsConsistent with previous studies, serum calprotectin level was revealed to have potential as a rapid diagnostic biomarker of disease activity in patients with SLE.

Lipoprotein capture ELISA method for the sensitive detection of amphiphilic biomarkers

Enzyme-linked immunosorbent assays (ELISAs) are widely employed for the detection of protein targets due to their ease of use, sensitivity, and potential for high-throughput analyses. However, the use of ELISAs to detect non-protein targets such as lipids and amphiphiles is complicated by the physical properties of these molecules, which affects their association with functional surfaces and recognition ligands. Here, we developed a unique lipoprotein capture ELISA in which the natural association between lipoproteins and amphiphilic molecules facilitates detection of the target biomarker in a physiologically relevant conformation. An assay to detect the glycolipid lipoarabinomannan (LAM), a cell membrane component and virulence factor associated with Mycobacterial infections, was developed as a proof of concept.

Generation and Characterization of an Anti-diclazuril Monoclonal Antibody and Development of a Diagnostic Enzyme-Linked Immunosorbent Assay for Poultry.

An anti-diclazuril monoclonal antibody (mAb) was developed for use in enzyme-linked immunosorbent assay (ELISA)-based detection of diclazuril with high sensitivity and specificity, which can be used to measure anti-coccidial drug residues. The anti-diclazuril mAb had a half-maximal inhibitory concentration of 0.449–0.517 ng/mL. The mAb cross-reactivity with toltrazuril, toltrazuril 18 sulfone, clozaril, monesin, madurmycin, and salinomycin was very minimal (< 0.1%). The detection limit of the ELISA using this mAb was 0.10 ng/mL and the sensitivity was 0.05 ng/mL. A standard curve generated in the range of 0.05–16.2 ng/mL had a linear correlation coefficient value of ≥ 0.99. The average recoveries of diclazuril from chicken and duck samples ranged from 85.0 to 102.5%.Intra- and inter-assay coefficients of variation ranged from 5.9 to 8.5% and 9.2 to 12.6%, respectively. Using the International Immunogenetics Information System®, the VH domain of the mAb was found to be encoded by an IGHV3 family gene and had the following complementarity determining region (CDR) sequences: GFTFSRY (CDR1), SRGGS (CDR2), and GDDNYAFAY (CDR3). The VL domain was encoded by an IGKV1 family gene and had the following CDR sequences: KSSQSLLNSRTRKNYLA (CDR1), WASTRES (CDR2), and KQSYNLHT (CDR3). This study provides a method to generate anti-diclazuril mAbs and determine their variable region sequences. The diagnostic ELISA developed using this mAb may drive additional studies on the monitoring and detection of food and veterinary drug residues.

Diagnosis of Coxiella burnetii Cattle Abortion: A One-Year Observational Study.

Q fever is a zoonosis occurring worldwide in livestock. Often neglected in differential diagnoses, Q fever can persist in herds causing financial losses. In ruminants, well-known manifestations of Q fever are metritis, infertility, abortion, stillbirth and delivery of a weak or premature calf. In cattle, Q fever is frequently asymptomatic and/or under-reported. Few studies are available on the diagnosis of Coxiella burnetii as a cause of abortion in cattle using polymerase chain reaction (PCR) for pathogen detection while enzyme-linked immunosorbent assay (ELISA) is used to assess exposure. Moreover, existing studies include a relatively small number of abortions. The aim of this study is to assess, in the southern part of Belgium, during a year, the performance of diagnosis of C. burnetii as a cause of abortion and the putative benefit of enhanced serology using anamnesis (animal patient data, and present, past and environmental history). A one-year random selection of 1212 abortions was analysed both with the PCR method (tissues from fetuses) and two commercialised ELISAs (sera from the mothers). Relative sensitivity and specificity of the ELISA tests were assessed using PCR as the reference test. The prevalence of C. burnetii PCR positive was 8.5% (95% CI: 6.99–10.21). The diagnostic value of the ELISA tests was assessed using the area under the receiver operating characteristic curve (AUC-ROC). The sensitivity, specificity and AUC-ROC were similar for both ELISA tests. The diagnostic capacity of the ELISA was confirmed and slightly enhanced if anamnestic information was integrated with a unique scoring index system. A high negative predictive value was demonstrated and a significant reverse association between Ct values and a percentage of the ratio of the optical density between the sample and the positive control (ELISA A or ELISA B) enabling the use of ELISA as an exclusion diagnostic. This study is original by integrating the serological result and the anamnesis in a single index. It opens a new window in enhanced veterinary clinical decision-making.

Identification and validation of serum autoantibodies in children with B-cell acute lymphoblastic leukemia by serological proteome analysis

BackgroundB-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy of childhood. Even though significant progresses have been made in the treatment of B-ALL, some pediatric B-ALL have still poor prognosis. The identification of tumor autoantibodies may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteome analysis (SERPA) to screen serum autoantibodies of pediatric B-ALL, aiming to contribute to the early detection of B-ALL in children.MethodsThe total proteins from three pooled B-ALL cell lines (NALM-6, REH and BALL-1 cells) were separated using two-dimensional gel electrophoresis (2-DE), which was followed by Western blot by mixed serum samples from children with B-ALL (n=20) or healthy controls (n=20). We analyzed the images of 2-D gel and Western blot by PDQuest software, and then identified the spots of immune responses in B-ALL samples compared with those in control samples. The proteins from spots were identified using mass spectrometry (MS). The autoantibodies against alpha-enolase (α-enolase) and voltage-dependent anion-selective channel protein 1 (VDAC1) were further validated in sera from another 30 children with B-ALL and 25 normal individuals by the use of enzyme-linked immunosorbent assay (ELISA). The protein expression levels of the candidate antigens α-enolase and VDAC1 in B-ALL were thoroughly studied by immunohistochemical analysis.ResultsUtilizing the SERPA approach, α-enolase and VDAC1 were identified as candidate autoantigens in children with B-ALL. The frequencies of autoantibodies against α-enolase and VDAC1 in children with B-ALL were 27% and 23% by using ELISA analysis, respectively, which were significantly higher than those in normal controls (4% and 0, p<0.05). Immunohistochemical analysis showed the expression of α-enolase and VDAC1 was positive in 95% and 85% of B-ALL patients, respectively, but negative expression levels were showed in the control group.ConclusionsThis study incidated that α-enolase and VDAC1 may be the autoantigens associated with B-ALL. Therefore, α-enolase and VDAC1 autoantibodies may be the potential serological markers for children with B-ALL.

Значение иммуноферментного анализа в серологической диагностике бруцеллеза крупного рогатого скота

For the serological diagnosis of brucellosis in cattle in veterinary laboratories of the Russian Federation, one or several tests are used simultaneously. The sensitivity and specificity of the determination of antibodies to the pathogens of this disease using various methods varies significantly, none of the serological tests is suitable for all stages of the infectious process in brucellosis. ELISA is a modern serological test that has not found wide application in the state veterinary laboratories of the Russian Federation, although the number of studies conducted by this method increases annually. The use of enzyme-linked immunosorbent assay makes it possible to confirm the diagnosis of brucellosis at all stages of the infectious process and to identify more seropositive animals than classical serological methods. Among serological methods, enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) is the most sensitive, specific and easy-to-perform test. In accordance with the Order of the Ministry of Agriculture of the Russian Federation dated 08.09.2020 No. 533 «On approval of Veterinary rules for the implementation of preventive, diagnostic, restrictive and other measures, the establishment and cancellation of quarantine and other restrictions aimed at preventing the spread and elimination of foci of brucellosis (including infectious epididymitis of sheep)» the ELISA method is one of the confirmatory in the diagnosis of brucellosis. The data obtained as a result of the studies indicates that the results of ELISA coincide with the complex of classical serological methods (BRS – Bengal roses sample; AR – agglutination reaction; CFR – complement fixation reaction; RIH – reaction of indirect hemaggtination; IDR – immunodiffusion reaction) and a higher diagnostic sensitivity of the test.

Probing Inflammasome Activation in Atherosclerosis.

Atherosclerosis is driven by chronic inflammation in all stages of the disease. Inflammation is fueled by elevated levels of pro-inflammatory cytokines. Interleukins (IL) are cytokines of particular importance in atherosclerosis, due to their key involvement in various pro-atherogenic processes, including infiltration of immune cells to the lesion, stimulation of the production of other pro-inflammatory mediators by other sources, and generation of lipid laden foam cells, all of which contribute to plaque development and progression. Various stimuli that are abundant in atherosclerotic plaques, including oxidized low-density lipoprotein, cholesterol crystals and reactive oxygen species can trigger inflammasome activation. Importantly, activation of the nucleotide oligomerization domain leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome activates the caspase-1 protease and results in the generation and release of potent pro-inflammatory cytokines, IL-1β and IL-18. Both cytokines are influential in driving chronic inflammation and atherogenesis. This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) and Western blot to quantify these cytokines in cell supernatant and lysate respectively, after stimulating inflammasome activation in cultured cells.

Diagnostic Accuracy of COVID-19 Antibody Tests Authorized by FDA Philippines: A Systematic Review and Meta-Analysis

Introduction: Coronavirus Disease (COVID-19) is a highly infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which has infected many people all over the world. One of the best ways to lessen its spread is through early detection and diagnosis. Various serological tests are now being used as a surveillance tool in the detection of antibodies as a response to SARS-CoV-2. The aim of this study is to evaluate the diagnostic accuracy and performance of the available COVID-19 antibody tests authorized by the Food and Drug Administration (FDA) Philippines that make use of Enzyme-Linked Immunosorbent Assay (ELISA), Chemiluminescence Immunoassay (CLIA) and Lateral Flow Immunoassay (LFIA). Method: Complete published journal articles relevant to the diagnostic accuracy of the three antibody tests were collected using trusted medical journal search engines. The quality of journals was assessed using QUADAS-2 to determine the risk of bias and assess the applicability judgments of diagnostic accuracy studies. Forest plots were used to summarize the performance of LFIA, ELISA and CLIA according to their specificity and sensitivity in detecting various antibodies. Pooled sensitivity and specificity were also done using bivariate random-effects models with its log-likelihood, a corresponding chi-square test statistic, and area under the summary Receiver-Operating Characteristic curve to see the potential heterogeneity in the data and to assess the diagnostic accuracy of the COVID-19 antibody tests. Results: Bivariate random-effects model and areas under the sROC curve were used to evaluate the diagnostic accuracy of COVID-19 antibody tests. The pooled sensitivity in detecting IgG based on CLIA, ELISA, and LFIA were 81.7%, 58.7%, and 74.3% respectively, with an overall of 72.0%. For IgM detection, LFIA has a higher pooled sensitivity of 69.6% than CLIA with 61.0%. Overall, the pooled sensitivity is 68.5%. In IgA detection, only ELISA based test was included with a pooled sensitivity of 84.8%. Lastly, pooled sensitivities for combined antibodies based on ELISA and LFIA were 89.0% and 81.6% respectively, with an overall of 82.5%. On the other hand, all tests excluding ELISA-IgA displayed high pooled specificities with a range of 94.0% to 100.0%. Diagnostic accuracies of the test in detecting IgG, IgM, and combined antibodies were found out to be almost perfect based on the computed area under the sROC with values of 0.973, 0.953, and 0.966, respectively. Conclusion: In this systematic review and meta-analysis, existing evidence on the diagnostic accuracy of antibody tests for COVID-19 were found to be characterized by high risks of bias, consistency in the heterogeneity of sensitivities, and consistency in the homogeneity of high specificities except in IgA detection using ELISA. The bivariate random-effects models showed that there are no significant differences in terms of sensitivity among CLIA, ELISA and LFIA in detecting IgG, IgM, and combined antibodies at a 95% confidence interval. Nonetheless, CLIA, ELISA and LFIA were found to have excellent diagnostic accuracies in the detection of IgG, IgM and combined antibodies as reflected by their AUC values. Doi: 10.28991/SciMedJ-2021-0304-1 Full Text: PDF

Seroprevalence and Risk Factors Associated with Canine Leishmaniasis in Egypt.

Background: Canine leishmaniasis (CanL) is caused by Leishmania infantum (L. infantum) that is transmitted by sand fly vectors with dogs acting as the main reservoir. Methods: The present study aimed to determine the seroprevalence of CanL in dogs from Egypt and assessed the associated risk factors. The study was conducted from 2019 to 2020 in five governorates situated in Northern Egypt. Serum samples from 450 asymptomatic dogs were serologically examined by use of enzyme-linked immunosorbent assay (ELISA). Results: Overall, the seroprevalence rate of CanL was 21.3% and the highest rates were observed in Cairo and Giza governorates. The univariable analysis revealed that the seropositivity of CanL was strongly related to the dogs’ ages, length of hair, absence of veterinary care or application of insecticides, and the type of floor of their shelters. The risk factors that were found to be associated with CanL in exposed dogs were: age group 2–4 years old (OR = 12, 95% CI: 1.6–92.3); short hair (OR = 2.07, 95% CI: 1.2–3.6); absence of veterinary care (OR = 2.7, 95% CI: 1.3–5.8); no application of insecticides (OR = 3.09, 95% CI: 1.5–6.5) and their residence in a shelter with an earthen floor (OR = 1.42, 95% CI: 0.7–2.9). Conclusions: Based on the present results, CanL is present in Egyptian dogs and this increases the possibility of transmission by sand fly to humans with whom they have contact. Consequently, an efficient monitoring programme and effective control measures are important to reduce the risk of infection.

S100A9-containing serum exosomes of burn injury patients promote permeability of pulmonary microvascular endothelial cells

Microvascular hyperpermeability is a leading mechanism responsible for occurrence of edema in remote organs and tissues in patients with burn injury. Accumulated evidence has shown that exosomes can be transported into target cells, where they are capable of regulating biological functions and physiology. Of exosomal proteins contributing to enhanced inflammation and vascular permeability, S100 calcium binding protein A9 (S100A9) has received increasing attention. Here we hypothesized that S100A9-containing serum exosomes of patients with burn injury contribute to pathogenesis of hyperpermeability of microvascular structure in lung by transferring signaling molecules into it and activating downstream signaling pathways, ultimately leading to disruption of the tight junctions (TJs) and endothelial barrier. A use of enzyme-linked immunosorbent assay revealed that total serum concentrations of S100A9 were significantly augmented in burn injury patients in comparison to normal controls. With use of human pulmonary microvascular endothelial cells (HPMECs) as an in vitro model, we found that patients' serum exosomes were effectively internalized by HPMECs. We further found that serum exosomes of stage II/II burn patients inhibited zonula occludens (ZO-1) and occludin protein levels, which are essential for TJs integrity and endothelial barrier function, but activated p38 MAPK signaling pathway in HPMECs. As expected, such exosomes-mediated effects on HPMECs were reversed by a simultaneous treatment of anti-S100A9 neutralizing antibody. Finally, we found that a recombinant human S100A9 treatment led to inhibition of expression of occludin and ZO-1 but an activation of p38 signaling in HPMECs, and that such effects were reversed when p38 activity was repressed, implying that S100A9 may stimulate p38 activity to inhibit ZO-1 and occludin in HPMECs. Collectively, these data suggest that S100A9-containing serum exosomes may play a critical role in contributing to pulmonary microvascular hyperpermeability, thus supporting that blocking exosomes' access to HPMECs could hold a promise strategy for treatment of lung edema resulting from burn injuries.

The application of immuno-enzymal analysis as a method of early express diagnostics of rednoya in pond fish

Due to the lack of effective means of specific prevention of rubella in pond fish (CRF), it is obvious that early diagnosis is one of the most important and effective ways to combat this disease. This theoretical study is devoted to the development of an express method for the rapid detection of antibodies to rubella pathogens in pond fish (Aeromonas punctata, Pseudomonas, Rabdovirus cyprini). To date, the diagnosis of CRC (Rabdovirus cyprini) is determined by the results of the clinical picture, taking into account anatomical data, epizootological indicators and virological research results. The diagnosis of CRC (Aeromonas punctata, Pseudomonas) is determined by bacteriological results, taking into account epizootological indicators, clinical signs and pathological changes. The use of enzyme-linked immunosorbent assay (ELISA) for the determination of CRC antibodies is a comprehensive diagnostic kit that allows you to significantly reduce the time for examination (sample preparation, reaction, results recording), and also reduce the number of errors caused by the human factor. The ELISA reaction makes it possible to quickly and efficiently obtain research results. As a result of the study, indicators of the effectiveness of the application of this technique for the diagnosis of CRC were proposed. The advantage of ELISA in comparison with the bacteriological method lies in the simplicity of setting and the reduction in the time of analysis (within 1-2 hours). The proposed method for the determination of rubella in pond fish using enzyme-linked immunosorbent assay allows, within the framework of laboratory monitoring, to carry out an express diagnosis of the CRC disease. The possibility of early diagnosis of this disease, the low cost of diagnostic kits and the ability to automate most stages of the reaction, demonstrate the practical value of the presented method.