Exploring the Association of VEGF-A and ANGPTL2 with the Prognosis of Non-proliferative and Proliferative Diabetic Retinopathy.

Introduction Diabetic retinopathy (DR) is a microvascular ailment that can arise from the long-term effects of diabetes mellitus. It can potentially cause retinal damage that could endanger vision and cause blindness. The worsening of DR is mainly linked to poor glycemic control, uncontrolled hypertension, and dyslipidemia. There is a need for alternative and clinically significant novel molecules involved in the pathogenesis of DR because the diagnostic and prognostic markers have reached a limit. Materials and method This study included sex and age-matched diabetic patients with proliferative stage (N = 70), non-proliferative stage (N = 80), and control (N = 80, without the sign of DR). These patients were recruited from outpatients in the Department of Ophthalmology, Sri Ramachandra Institute of Higher Education and Research, Chennai, India. A random blood sample was collected from each study participant, and the serum was separated after centrifugation and stored at -80 °C for batch analysis. The biomarkers vascular endothelial growth factor (VEGF-A) and angiopoietin-like protein-2 (ANGPTL2) were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) technique, and the laboratory parameters such as fasting blood sugar (FBS), lipid profile, blood urea nitrogen (BUN), creatine, and glycated hemoglobin (HbA1C) were also assessed. Results We observed statistically significant differences in the duration of diabetes, FBS, total cholesterol (TC), triglyceride level (TGL), BUN, and creatine (p<0.05), and the mean age of study participants was 52.95±8.20 years in the control group, 53.85±10.20 years in the proliferative diabetic retinopathy (PDR) group, and 55.02±7.65 in the non-proliferative diabetic retinopathy (NPDR) group. Furthermore, ANGPTL2 levels were statistically significant according to the severity of the disease (p<0.001*), and they were also linked (p<0.05) with established markers such as VEGF-A. Conclusion Thus, our research implies that the up-regulated markers might be linked to the disease's advancement and could serve as a prognostic indicator or therapeutic target for DR.

Performance of post-mortem diagnostic tests for tuberculosis in wild ungulates at low and high prevalence assessed using Bayesian latent class models.

Animal tuberculosis (TB) is often maintained by multi-host communities, including livestock and wildlife. Quantitative studies of such communities require estimating the true prevalence of TB, correcting the apparent prevalence by the diagnostic sensitivity (Se) and specificity (Sp) of the test. The goal of this study was to lay the foundations for estimating the true prevalence of TB in wild ungulate populations (wild boar and two cervids: red deer and fallow deer). We used Bayesian latent class models to assess the Se and Sp of gross pathology, IS6110 real-time PCR in tissues, bacteriological culture, and P22 indirect ELISA. We analyzed 308 harvested wild ungulates (211 wild boar and 97 cervids: 92 red deer and 5 fallow deer). The Se of bacteriological culture (80.4%, CI95 61.0-96.3%) and gross pathology (87.9%, CI95 69.5-99.9%) was reasonably good in wild boar. These tests showed lower Se in cervids: 60.2% (CI95 38.3-82.3%) for bacteriological culture and 81.5% (CI95 63.6-96.2%) for gross pathology. The Se of the real-time PCR was low (50.7% in wild boar and 53.0% in cervids). These tests showed Sp between 95.2 and 99.1% in both taxa. The P22 ELISA performed reasonably well in wild boar (Se = 71.9%, CI95 59.2-83.4%; Sp = 98.8%, CI95 96.9-99.9%) but lacked Sp in cervids (Se = 77.1%, CI95 62.9-89.7%; Sp = 74.5%, CI95 65.7-83.3%). The real-time PCR in wild boar and cervids and bacteriological culture in cervids tended to show higher Se in low-prevalence populations, possibly due to a higher proportion of early-stage TB lesions. In cervids, the parallel interpretation of gross pathology and bacteriological culture significantly improved the diagnostic performance (Se = 93.1%, CI95 84.7-98.9%; Sp = 92.9%, CI95 86.0-98.3%). Our results allow the estimation of true prevalence from the results of a single diagnostic test applied to harvested wild boar, red deer, and fallow deer, paving the way for more precise quantitative ecological studies of the multi-host TB maintenance community.

The effect of periodontal health and disease on interleukin 1ß and nesfatin-1 levels in gingival crevicular fluid: A cross-sectional study.

Introduction: Periodontitis, a chronic inflammatory disease associated with dental biofilm, poses a significant threat to oral health. This study explores the roles of interleukin 1ß (IL-1ß) and nesfatin-1 in periodontal diseases, aiming to contribute to the molecular understanding of their pathogenesis. Material and methods: A diverse cohort of 62 participants was recruited, spanning ages 20 to 60, and categorized into healthy, gingivitis, and periodontitis groups. Clinical measurements, including plaque index, gingival index, probing pocket depth, and bleeding on probing, were conducted. Gingival crevicular fluid (GCF) samples were collected for IL-1ß and nesfatin-1 analysis using enzyme-linked immunosorbent assay (ELISA). Statistical analysis employed Kruskal-Wallis and Spearman correlation tests. Results: Significant differences in oral hygiene habits were observed among groups, particularly in the 40-60 age range. Clinical indices showed variations, with the highest IL-1ß levels in the periodontitis group and the lowest nesfatin-1 levels. Correlation analysis revealed positive associations between IL-1ß, nesfatin-1, and oral indices. Conclusions: While providing valuable insights, we acknowledge this study's limitations, including a cross-sectional design and a specific age range. Future research should employ longitudinal designs and larger cohorts, and explore broader inflammatory markers, genetic influences, and confounding variables for a more comprehensive understanding of periodontal diseases. The findings underscore the complex interplay between inflammatory markers and periodontal health.

ELISA genotyping of hepatitis B virus in China with antibodies specific for genotypes B and C.

Hepatitis B virus (HBV) causes hepatitis B (HB) and distinct HBV genotypes can lead to different prognoses. However, HBV genotyping is rarely done in clinics, because the traditional method by PCR-based DNA sequencing is impractical for clinical diagnosis with tedious process and low success rate. Herein, we have established an ELISA-based genotyping method to quickly determine the HBV genotypes of HB patients in China. First, two commercial antibodies, 16D12 and 6H3 specific for HBV genotypes B and C respectively, are chosen as capture antibodies, since these two genotypes dominate in China. Then two home-made genotype-specific antibodies, B19 and C04, are used as the detection antibodies for genotypes B and C in sandwiched ELISA. The ELISA kit shows high sensitivity (> 95%) and specificity (> 95%) in detecting genotypes B and C of Chinese HB patients. Moreover, the ELISA kit has demonstrated higher success rate (98.7%) than PCR-based DNA sequencing (93.5%) and a commercial PCR-based genotyping kit (92.2%) for sera with HBV DNA ≥ 1000 IU/mL and HBsAg ≥ 250 IU/mL. Such an advantage is more obvious for the sera with HBV DNA < 1000 IU/mL. The kappa analysis between the ELISA and PCR-based DNA sequencing results exhibits a kappa of 0.836, indicating a good correlation.

Plasma neurofilament heavy chain is a prognostic biomarker for the development of severe epilepsy after experimental traumatic brain injury.

OBJECTIVE: This study was undertaken to test whether the postinjury plasma concentration of phosphorylated neurofilament heavy chain (pNF-H), a marker of axonal injury, is a prognostic biomarker for the development of posttraumatic epilepsy. METHODS: Tail vein plasma was sampled 48 h after traumatic brain injury (TBI) from 143 rats (10 naïve, 21 controls, 112 with lateral fluid percussion injury) to quantify pNF-H by enzyme-linked immunosorbent assay. During the 6th postinjury month, rats underwent 30 days of continuous video-electroencephalographic monitoring to detect unprovoked seizures and evaluate epilepsy severity. Somatomotor (composite neuroscore) and spatial memory (Morris water maze) testing and quantitative T2 magnetic resonance imaging were performed to assess comorbidities and lesion severity. RESULTS: Of the 112 TBI rats, 25% (28/112) developed epilepsy (TBI+) and 75% (84/112) did not (TBI-). Plasma pNF-H concentrations were higher in TBI+ rats than in TBI- rats (p < .05). Receiver operating characteristic curve analysis indicated that plasma pNF-H concentration distinguished TBI+ rats from TBI- rats (area under the curve [AUC] = .647, p < .05). Differentiation was stronger when comparing TBI+ rats exhibiting severe epilepsy (≥3 seizures/month) with all other TBI rats (AUC = .732, p < .01). Plasma pNF-H concentration on day 2 (D2) distinguished TBI+ rats with seizure clusters from other TBI rats (AUC = .732, p < .05). Higher plasma pNF-H concentration on D2 after TBI correlated with lower neuroscores on D2 (p < .001), D6 (p < .001), and D14 (p < .01). Higher pNF-H concentration on D2 correlated with greater T2 signal abnormality volume on D2 (p < .001) and D7 (p < .01) and larger cortical lesion area on D182 (p < .01). Plasma pNF-H concentration on D2 did not correlate with Morris water maze performance on D37-D39. SIGNIFICANCE: Plasma pNF-H is a promising clinically translatable prognostic biomarker for the development of posttraumatic epilepsy with frequent seizures or seizure clusters.

Development of sandwich enzyme-linked immunosorbent assays quantifying mouse urinary megalin, a novel proximal tubular biomarker.

Megalin, a type I transmembrane protein, serves as a multi-ligand endocytic receptor in the apical membrane of proximal tubules. Its ectodomain and full-length forms are excreted into human urine, with the former being more abundant. We previously developed two types of sandwich enzyme-linked immunosorbent assays (ELISAs) utilizing monoclonal antibodies that target the amino-terminal ligand-binding domain-I and the carboxyl-terminal cytoplasmic region of human megalin, respectively. The former, termed "A-megalin" ELISA, primarily identifies ectodomains of megalin, whereas the latter, "C-megalin" ELISA, specifically recognizes full-length megalin originating from urinary extracellular vesicles. This study developed novel sandwich ELISAs to assess mouse urinary A-megalin and C-megalin, thereby facilitating studies involving these biomarkers in mouse disease models. Immunoblotting and immunohistochemistry of monoclonal antibodies against human megalin were performed to assess their compatibility with mouse megalin in novel sandwich ELISAs, which were constructed and validated using human assay protocols. Immunoblot analysis of megalin in urinary extracellular vesicles and supernatant was performed to investigate the ratio of ectodomain to full-length forms in mouse urine. Stable measurements having a precision and accuracy within 15 % were achieved in the measurement of quality control samples. A-megalin and C-megalin were detectable in the urine of C57BL/6 mice, whereas most urine samples from kidney-specific conditional megalin-knockout mice were below detection limits. Ectodomain forms of megalin were at least approximately 70 times more abundant than the full-length form, even in mouse urine. In conclusion, we successfully developed sandwich ELISAs for assessing mouse urinary A-megalin and C-megalin to evaluate primarily ectodomain and full-length forms of megalin, respectively.

Comparison of protein immobilization methods with covalent bonding on paper for paper-based enzyme-linked immunosorbent assay.

In this study, two methods were examined to optimize the immobilization of antibodies on paper when conducting a paper-based enzyme-linked immunosorbent assay (P-ELISA). Human IgG, as a test-capture protein, was immobilized on paper via the formation of Schiff bases. Aldehyde groups were introduced onto the surface of the paper via two methods: NaIO4 and 3-aminopropyltriethoxysilane (APTS) with glutaraldehyde (APTS-glutaraldehyde). In the assay, horseradish peroxidase-conjugated anti-human IgG (HRP-anti-IgG) binds to the immobilized human IgG, and the colorimetric reaction of 3,3',5,5'-tetramethylbenzyzine (TMB) produces a blue color in the presence of H2O2 and HRP-anti-IgG as a model analyte. The immobilization of human IgG, the enzymatic reaction conditions, and the reduction of the chemical bond between the paper surface and immobilized human IgG all were optimized in order to improve both the analytical performance and the stability. In addition, the thickness of the paper was examined to stabilize the analytical signal. Consequently, the APTS-glutaraldehyde method was superior to the NaIO4 method in terms of sensitivity and reproducibility. Conversely, the reduction of imine to amine with NaBH4 proved to exert only minimal influence on sensitivity and stability, although it tended to degrade reproducibility. We also found that thick paper was preferential when using P-ELISA because a rigid paper substrate prevents distortion of the paper surface that is often caused by repeated washing processes.

A novel recombinant Theileria annulata surface protein as an antigen in indirect enzyme-linked immunosorbent assay for the serological diagnosis of tropical theileriosis.

Background and Aim: Theileria annulata infection in cattle causes major economic losses in livestock production in many Central Asian countries, including the southern region of Kazakhstan. This study aimed to obtain a recombinant T. annulata surface protein (TaSP) and to investigate its possible use as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of bovine theileriosis. Materials and Methods: Recombinant TaSP was obtained by cloning a polymorphic region of the TaSP gene, expressing it in Escherichia coli strain BL21, and purifying it by metal chelating chromatography. An indirect ELISA using recombinant TaSP as an antigen was developed and evaluated for the detection of T. annulata-specific antibodies in plasma samples from 69 cows polymerase chain reaction (PCR)-positive or PCR-negative for T. annulata and/or Theileria orientalis from southern Kazakhstan. Results: The obtained recombinant protein had a molecular weight of 32 kDa, and mass spectrometry analysis of the purified protein identified it as a fragment of the surface protein of T. annulata. Initial testing of 69 field plasma samples from cattle showed that the results of indirect ELISA using TaSP as an antigen agreed substantially with those of T. annulata PCR (κ: 0.78). The relative sensitivity and specificity of indirect ELISA were 88.7% and 100%, respectively, using PCR as a reference. There was no evidence of cross-reaction with T. orientalis. Conclusion: Initial results using recombinant TaSP as an antigen in indirect ELISA are promising and support the widespread use of this assay for routine diagnosis and T. annulata seroprevalence studies in cattle in Kazakhstan and possibly neighboring countries.

Establishment of a Quenchbody-based L-thyroxine detection method and its comparison with ELISA systems.

The quantification of L-thyroxine (T4) is crucial for regulating metabolism, diagnosing diseases, and monitoring the efficacy of T4 replacement therapy. However, because T4 is a hapten biomarker with a molecular weight of 777 g/mol, conventional immunoassay approaches, including Western blotting and some types of ELISA, have limited accuracy in the quantification of small molecules, including T4. Furthermore, these methods are time-consuming and involve multiple incubation and reaction steps. Therefore, a novel immunoassay method is required for simple and rapid on-site detection of T4. In this study, we expressed a recombinant anti-T4 single-chain variable fragment (scFv) in soluble form using Escherichia coli. The scFv exhibited high T4-binding efficiency, and T4 concentration-dependent titration curves indicated that the sandwich ELISA could detect T4 in the nanogram range. We labeled the scFv using a fluorescent dye for a Quenchbody (Q-body)-based one-pot immunoassay, which yielded a T4 concentration-dependent fluorescent response in 3 min. A comparison of the Q-body-based T4 detection system with ELISA-based methods demonstrated that the ELISA system was more sensitive but the Q-body assay was more rapid. Therefore, both ELISA and Q-body systems can be used depending on the experimental purpose, with the newly developed anti-T4 Q-body system being applicable for convenient in situ immunoassay of T4.

Circulating Interleukins as Biomarkers in Non-Small Cell Lung Cancer Patients: A Pilot Study Compared to Normal Individuals.

Identifying biomarkers in non-small cell lung cancer (NSCLC) can improve diagnosis and patient stratification. We evaluated plasmas and sera for interleukins (IL)-11, IL-6, IL-8, IL-17A, and IL-33 as biomarkers in primary NSCLC patients undergoing surgical treatment against normal volunteers. Exhaled-breath condensates (EBCs), a potential source without invasive procedures, were explored in normal individuals. Due to separate recruitment criteria and intrinsic cohort differences, the NSCLC and control cohorts were not well matched for age (median age: 65 vs. 40 years; p < 0.0001) and smoking status (p = 0.0058). Interleukins were first assessed through conventional ELISA. IL-11 was elevated in NSCLC plasma compared to controls (49.71 ± 16.90 vs. 27.67 ± 14.06 pg/mL, respectively, p < 0.0001) but undetectable in sera and EBCs by conventional ELISA. Therefore, high-sensitivity PCR-based IL-11 ELISA was repeated, albeit with concentration discrepancies. IL11 gene and protein upregulation by RT-qPCR and immunohistochemistry, respectively, were validated in NSCLC tumors. The lack of detection sensitivity across IL-6, IL-8, IL-17A, and IL-33 suggests the need for further, precise assays. Surprisingly, biomarker concentrations can be dissimilar across paired plasmas and sera. Our results identified a need to optimize detection limits for biomarker detection and caution against over-reliance on just one form of blood sample for biomarker assessment.

Seroprevalence and Associated Risk Factors of Infectious Bovine Rhinotracheitis (IBR) and Animal Owners' Knowledge, Attitude and Practice (KAP) Towards the Disease in Selected Districts of East Wollega Zone, Oromia Regional State, Ethiopia.

PURPOSE: Infectious bovine rhinotracheitis (IBR) is one of the most important diseases affecting production and productivity. METHODOLOGY: Cross-sectional study was aimed at to determine the seroprevalence of IBR and associated risk factors, and animal owners' knowledge, attitude and practice towards the disease from April 2021 to June 2022. Accordingly, a total of 384 serum samples were collected from both crossbreed (70) and local breed (314) cattle from purposively selected districts of East Wollega zone of Western Ethiopia. Competitive enzyme-linked immunosorbent assay (ELISA) was used for testing glycoprotein antibodies (anti-gB) for bovine herpes virus-1 (BoHV-1) virus in collected serum, and the obtained data were analysed by multiple logistic regressions by using R software 3.62 version. However, questionnaire data were analysed for descriptive statistics by SPSS version 20.0 (IBM. Corp, 2011). RESULT: The total prevalence of IBR in the study area was found to be 70.54% at herd and 80.47% at individual cattle level. The significant association (p  < 0.05 $ < \ 0.05$ ) was found for breed, age, body condition and herd size but not for district and sex as risk factors. The BoHV-1 virus seropositivity in adult animals increased significantly, with an odds ratio of 1.65 (95% CI 0.705-3.85) compared to young. Local breed cattle were 2.055 times more likely to test positive for IBR with an odds ratio of 0.77 (95% CI 0.23-2.22) compared to crossbreed cattle. The chances of cattle in medium herds testing positive for the BoHV-1 virus with an odds ratio of (1.78 95% CI 1.303-7.50) are greater than the chances of cattle in smaller herds testing positive. The survey results showed that 70% of animal owners identified IBR as a major challenge in animal production, whereas 35% mentioned long calving intervals. However, 92% of the participants were not informed about the level of knowledge and attitude regarding particular diseases such as IBR. CONCLUSION: This study showed that there is a high prevalence of IBR in cattle in the study area, and that owners have low awareness of the disease. Therefore, it is necessary to develop an immediate control system and conduct additional research on molecular detection to evaluate its effects on reproductive performance.

Targeting Design of Human Anti-idiotypic Genetically Engineered Antibody for Simulating the Structure and Insecticidal Function of Bt Cry1C Toxin.

The ß-type anti-Id (Ab2ß) is considered to have potential for simulating the structure and function of the antigen. In this study, a ß-type anti-Id (3A7 anti-I-GEAb) of the Cry1C toxin was captured from a GEAb library. Subsequently, a higher activity of mutant (3A7 mutant 8) was obtained from the mutagenesis library based on 3A7 anti-I-GEAb. The LD50 values of 3A7 anti-I-GEAb and 3A7 mutant 8 reach up to 38.9% and 46.8% of Cry1C toxin for P. xylostella and reach up to 32.9% and 37.4% of Cry1C toxin for H. armigera. Additionally, an IC-ELISA was established based on 3A7 mutant 8 (as the coated "antigen"), with an LOD value of 0.35 ng/mL, exhibiting good accuracy and stability for detecting Cry1C toxin in spiked samples. The present ß-type anti-I-GEAb not only exhibits insecticidal activity similar to Cry1C toxin, offering potential for environmentally friendly pest management, but it can also replace the Cry1C toxin structure to establish a highly sensitive and specific IC-ELISA for monitoring Cry1C toxin.

Prevalence, Clinico-Laboratory Features and Outcomes of Paediatric Scrub Typhus Cases in a Tertiary Care Centre in Eastern India: A prospective observational study.

Objectives: Scrub typhus is the most common rickettsial disease in India, caused by Orientia tsutsugamushi and transmitted by chigger mites. Previously prevalent in South India, a resurgence of scrub typhus cases has recently affected Eastern India. This study aimed to estimate the prevalence and describe the clinico-laboratory profile of scrub typhus in paediatric patients (1-12 years old) living in Eastern India. Methods: This prospective observational study was conducted from January to December 2019 at the Dr B C Roy Post Graduate Institute of Paediatric Sciences, Kolkata, India. All acute undifferentiated cases of febrile illness, in patients aged between 1-12 years, were tested using scrub typhus serology by ELISA. Demographic details, clinical features, laboratory findings, complications and treatment outcomes of these scrub typhus patients were extracted and analysed. Results: Out of 1,473 patients with acute febrile illness, 67 (4.5%) children were diagnosed with scrub typhus. The mean age of the selected patients was 5.22 ± 3.05 years, and the majority (64.2%) had been running a fever since the preceding 7-14 days. Gastrointestinal symptoms such as vomiting (43.3%) and abdominal pain (32.8%) were most frequently observed. Major clinical signs of scrub typhus were hepatomegaly (41.8%) and splenomegaly (31.3%). Complications were observed in 74.6% of patients, with thrombocytopenia (40.3%) and meningoencephalitis (29.9%) occurring more frequently. The case fatality rate of the study sample was 1.5%. Conclusion: Classical eschar was absent in three-fourth of the studied patients. Hence, this study advocates laboratory scrub typhus tests for all suspected cases in the endemic region (Eastern India). Prompt treatment with doxycycline and/or azithromycin could prevent complications such as thrombocytopenia/meningoencephalitis and reduce mortality.

Development of an ic-CLEIA for precise detection of 3-CQA in herbs and patent medicines: ensuring quality control and therapeutic efficacy.

Background: 3-caffeoylquinic acid (3-CQA), a member of the chlorogenic acid family, possesses diverse pharmacological properties, such as scavenging, antioxidant, and antiapoptotic activity, rendering substantial value to alimentary consumables and therapeutic substances. However, the pervasiveness of non-standard practices, notably the misuse and abuse of indigenous botanicals, coupled with the inherent susceptibility of 3-CQA to degradation under light and heat exposure, engenders discernible disparateness in the quality profiles of the same kinds of herbs. Consequently, precise quantification of 3-CQA becomes imperative. Methods: In this context, an artificial antigen was synthesized as a specific conjugate of 3-CQA and bovine serum albumin (3-CQA-BSA), followed by the generation of a monoclonal antibody (mAb) against the conjugate. Through optimization, a mAb-based indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) was developed. Results: It demonstrated an IC50 and the calibration range of 2.97 ng/mL and 0.64-13.75 ng/mL, respectively, outperforming the conventional enzyme-linked immunosorbent assay (ELISA). Notably, the ic-CLEIA displayed 10.71% cross-reactivity with 3,5-dicaffeoylquinic acid, alongside minimal cross-reactivity toward other isomeric counterparts and analogs. Validation experiments on herbs and Chinese patent medicines using ic-CLEIA, confirmed by high-performance liquid chromatography (HPLC) analysis, revealed a robust correlation coefficient of 0.9667 between the two modalities. Conclusion: These findings unequivocally demonstrated that the proposed ic-CLEIA represents a viable and reliable analytical method for 3-CQA determination. This method holds significant potential for ensuring the quality control and therapeutic efficacy germane to herbs and patent medicines, spanning diverse therapeutic milieus and applications.

Peanut-specific IgG subclasses as biomarkers of peanut allergy in LEAP study participants.

Antigen-specific IgG2 and IgG3 are rarely measured in food allergy clinical trials despite known function in preventing mast cell and basophil activation. Our objective was to determine whether measuring peanut-specific IgG2 and IgG3 levels would correlate with peanut allergy status. Peanut-specific IgG subclasses were measured via ELISA assays in Learning Early About Peanut allergy (LEAP) trial participants at 5 years of age and were correlated with peanut allergy vs peanut sensitization vs non-peanut allergic and peanut consumption vs peanut avoidance. Peanut-specific IgG1, IgG2, IgG3, and IgG4 levels were significantly different between participants with peanut allergy vs peanut sensitization vs non-peanut allergic, and a multivariate logistic regression model and stepwise selection found that IgG1 most closely associated with peanut allergy status. Similarly, all subclasses differentiated those consuming vs those avoiding peanut, but subsequent modeling found that IgG4 most closely associated with consumption status. Amongst the peanut-specific IgG subclasses, IgG1 was the best biomarker for peanut allergy, while IgG4 was the best biomarker for peanut antigen exposure in this highly atopic cohort. Our study did not find added value from evaluating peanut-specific IgG 2 and 3 as biomarkers of peanut allergy, although they did correlate with peanut allergy. Subsequent studies should assess the value of adding IgG subclasses to multivariate models predicting peanut allergy status.

A comparative study of anti-ADAMTS-13 antibody dynamics in immune-mediated thrombotic thrombocytopenic purpura.

Background: Thrombotic thrombocytopenic purpura, particularly its immune-mediated variant (iTTP), necessitates accurate diagnostic approaches for effective management. Objectives: To compare a chemiluminescence immunoassay (CLIA) and an enzyme-linked immunosorbent assay (ELISA) for testing ADAMTS-13 activity and detecting anti-ADAMTS-13 autoantibodies (AAbs) in patients with iTTP. Methods: This study involved 31 paired samples from 12 iTTP patients. ADAMTS-13 activity was measured using the HemosIL AcuStar (Instrumentation Laboratory, CLIA) and Technozym (Technoclone) activity assay (ELISA). The presence of AAbs was assessed using Technozym ADAMTS-13-INH assay (ELISA) and HemosIL AcuStar activity (CLIA) within a Bethesda assay following mixing with normal pool plasma. von Willebrand factor (VWF) multimers were analyzed using the HYDRASYS-2 SCAN system and the HYDRAGEL 5- or 11-VW Multimer kits (Sebia). VWF activity levels were measured with the HemosIL AcuStar VWF:GPIbR on the ACL AcuStar Analyzer (IL). Results: For ADAMTS-13 activity, a strong linear relationship and no bias between CLIA and ELISA were confirmed (slope = 1.01 [0.91, 1.11], intercept = 0.00 [-0.47, 0]). However, significant discrepancies were found in AAb detection during remission phases with ADAMTS-13 activity between 10% and 50%, with CLIA and ELISA showing significant divergence (P < .001, Cohen's g = 0.34). Consistently, VWF multimers and activity levels exhibited significantly different values between remission samples with ADAMTS-13 activity below 50% and above 50%. In longitudinal analysis of patients with multiple iTTP relapses, positivity to CLIA appears to precede ELISA in predicting exacerbations. Conclusion: While CLIA and ELISA might be interchangeable for assessing ADAMTS-13 activity, they are not equivalent for detecting AAbs, particularly in patients in clinical remission with ADAMTS-13 activity between 10% and 50%.

Comparison of urine and serum IgG detection ELISA for tegumentary leishmaniasis diagnosis and prognosis.

Laboratorial diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests, which are still hampered by blood́ invasive collection. In this context, in the present study, we develop a serum- and urine-based ELISA to TL diagnoses. A recombinant protein (rLiHyA), which was previously showed to be antigenic for the disease, as well as a B-cell epitope produced as synthetic peptide and a Leishmania antigenic extract (SLA), were used as antigens. A total of paired 205 urine and serum samples were used, which were comprised by samples from cutaneous (n = 30) and mucosal (n = 30) leishmaniasis patients, as well as from healthy individuals living in endemic region of disease (n = 45), of patients with Chagas disease (n = 30), leprosy (n = 35), malaria (n = 15) or HIV-infected (n = 20). Results showed that serum-based ELISA presented sensitivity of 24.0 %, 100 % and 41.0 %, when SLA, rLiHyA and synthetic peptide were used as antigens, and specificity of 98.4 %, 98.4 % and 98.4 %, respectively. The area under the curve (AUC) was calculated and results were 0.74, 1.0, and 0.71, respectively, when SLA, rLiHyA and synthetic peptide were used as antigens. Performing an urine-based ELISA, sensitivity was 28.0 %, 100 % and 75.0 %, respectively, when SLA, rLiHyA, and synthetic peptide were used, while specificity values were of 98.4 %, 98.4 % and 98.4 %, respectively. In addition, the AUC values were 0.82, 1.0, and 0.94, respectively. A significant drop in specific antibodies levels in both patients serum and urine samples was found six months after treatment, suggesting a prognostic role of rLiHyA for TL. In conclusion, preliminary data suggest the potential of use patient urine to TL diagnoses.

Application of Recombinant Monoclonal Antibodies from Transgenic Chicken Bioreactors in Enzyme-Linked Immunosorbent Assay.

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.

A new perspective on essential tremor: the potential etiologic role of Toxoplasma gondii and Toxocara spp.

Our aim was to examine the relationship between Toxoplasma gondii (T. gondii) and Toxocara infection and patients with essential tremor (ET). This study comprised a total of 174 participants, consisting of 99 patients with ET and 75 healthy controls. The presence of anti-T. gondii IgG and anti-Toxocara IgG antibodies was investigated using ELISA. The relationship between the severity of the disease and the seropositivity of T. gondii and Toxocara were examined. The seropositivity rate for anti-T. gondii IgG antibodies among patients and control groups were 43.4% and 12%, respectively (odds ratio [OR]: 5.63; 95% CI: 2.53-12.56). The patient group exhibited a higher seroprevalence of anti-Toxocara IgG antibodies (32.3%) compared with the control group (13.3%; OR: 3.10; 95% CI: 1.41-6.83; p = 0.004). This study suggests that T. gondii and Toxocara infections can contribute to the pathogenic mechanisms underlying ET and could be risk factors for ET.

Recombinant outer membrane protein Lipl41 from Leptospira interrogans robust immune responses in mice model.

Background and Objectives: Leptospirosis is an infectious zoonotic disease that can result in severe complications. It is widespread, especially in hot and humid climates such as the northern region of Iran. The immune responses to leptospirosis are multifaceted. Lipl41 is an outer membrane protein that is expressed during infection and is highly conserved among pathogenic species. This makes it a good candidate for diagnosis and induction of specific immune responses. The aim of the present study was to evaluate immune responses against recombinant Lipl41 in mice. Materials and Methods: After immunizing of different groups of mice with recombinant Lipl41 (rLipl41), the levels of specific antibodies and cytokine profiles interferon-gamma/interleukin-4 (IFN-γ/IL-4) were measured. Results: The results revealed that rLipl41 showed a significant increase in antibody levels compared with the control groups (P< 0.05). Although the level of IL-4 in the groups that received Lipl41 was similar to that in the other control groups, the IFN-γ levels showed a significant increase (P<0.05). Conclusion: It has been concluded that recombinant Lipl41 protein could strongly stimulate specific immune responses and be considered a potential candidate for vaccine development and diagnostic research.