Circulation of foot-and-mouth disease serotypes, risk factors, and their effect on hematological and biochemical profiles among cattle and buffalo in Quetta, Balochistan, Pakistan.

Background and Aim: Foot-and-mouth disease (FMD) is an infectious disease of cloven-hoofed animals, including buffalo, cattle, sheep, goats, and pigs, causing major economic losses to the local farmers and, overall, to the national economy of the country. This study aimed to detect FMDV serotypes in year-round FMD outbreaks, hematological and biochemical changes, and oxidative stress in FMDV-infected cattle and buffaloes in the district of Quetta, Balochistan, Pakistan, and the socioeconomic impact of FMD outbreaks on farmers. Materials and Methods: We conducted a cross-sectional study in the district of Quetta, Balochistan, Pakistan, where FMD virus (FMDV) serotypes were detected by enzyme-linked immunosorbent assay (ELISA). Hematological, biochemical, and oxidative analyses were performed by analyzing the blood of FMDV-infected and non-infected animals. Information on the associated risk factors was obtained through a structured questionnaire by interviewing farmers in each FMD-affected farm. Results: Thirty-four out of 38 farms (89%, 95% confidence interval [CI]: 75%-97%) were positive for FMD by ELISA. Higher FMD infection was detected in farms with a herd size of <50 animals (50%, 17/34), followed by >100 animals (32%, 11/34) and 51-100 animals (18%, 6/34). Fifty-seven percent (114/200, 95% CI: 50%-64%) of animals were positive for FMD. Of these, 61% (69/114) were cattle and 39% (45/114) were buffalo. FMD positivity was higher in females (86%, 98/114) than in males (14%, 16/114) and higher in animals older than 2 years of age (52%, 59/114). On average, farmers lose U.S. dollars 3000 annually due to FMD outbreaks. Animals infected with FMDV had significantly (p ≤ 0.05) white blood cell counts and significantly (p ≤ 0.05) lower hemoglobin and total protein concentrations in buffalo and cattle, whereas infected cattle showed significantly (p ≤ 0.05) lower albumin levels. Globulin levels were lower in buffaloes infected. Alanine aminotransferase levels were lower in infected cattle (p ≤ 0.05). Creatinine levels were higher in infected buffalo (p ≤ 0.05). Urea and phosphorus concentrations were higher in FMDV-infected cattle and buffalo (p ≤ 0.05). Calcium levels were lower in infected cattle and buffalo (p ≤ 0.05). Catalase enzyme activity in infected cattle and buffaloes was significantly lower (p < 0.05). Lipid peroxidation was significantly higher in FMDV-infected cattle and buffalo (p ≤ 0.05). Conclusion: This study confirmed serotype O circulation among cattle and buffalo in year-long FMD outbreaks in the Quetta District of Balochistan. Blood analysis identified a parameter deviated from the normal level due to FMDV infection. In addition, the outbreak of FMD has a significant negative economic impact on livestock farmers.

Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay1.

Background/Objective: Myasthenia Gravis (MG) is an autoimmune disorder characterized by pathogenic autoantibodies (AAbs) targeting nicotinic acetylcholine receptors (AChR), disrupting neuromuscular communication. RadioImmunoPrecipitation Assay (RIPA) is recommended to detect AChR AAbs, but its complexity and radioactive requirements limit widespread use. We compare non-RIPA anti-AChR immunoassays, including Cell-Based Assay (CBA) and two ELISA kits, against the gold standard RIPA. Methods/Results: 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg < 0.02 nmol/L), 18 were classified as Borderline (≥0.02 -1 nmol/L), and 64 were positive (RIPA-Pos > 1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappa = 0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappa = 0.652), with ∼76% sensitivity and ∼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappa = 0.984), yielding ∼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (r = 0.793 and r = 0.789, respectively). Conclusions: The CBA displayed excellent analytical performance for MG diagnostic when compared to RIPA, making it a potential replacement for RIPA in clinical laboratories. Some solid-phase assays (such as the indirect ELISA applied here), as well as CBA titration, offer reliable options to estimate anti-AChR AAb levels after confirming positivity by the CBA.∥.

Expression and Function of Connexins in Extracellular Vesicles.

Extracellular vesicles (EVs) are recognized as major vehicles for exchange of information across distant cells and tissues, which have been extensively explored for diagnosis and therapeutic purposes. The presence of multiple connexin (Cx) proteins has been described in EVs, where they might facilitate EV-cell communication. However, quantitative changes in Cx levels and functional assessment of Cx channels have only been established for Cx43. In present work, we provide a detailed description of the protocols we have optimized to assess the expression and permeability of Cx43 channels in EVs derived from cultured cells and human peripheral blood. Particularly, we include some modifications to improve quantitative analysis of EV-Cx43 by enzyme-linked immunosorbent assay (ELISA) and assessment of channel functionality by sucrose-density gradient ultracentrifugation, which can be easily adapted to other Cx family members, leveraging the development of diagnostic and therapeutic applications based on Cx-containing EVs.

In vitro evaluation of the binding activity of novel mouse IgG1 opsonic monoclonal antibodies to Mycobacterium tuberculosis and other selected mycobacterial species.

Antimicrobial resistance alongside other challenges in tuberculosis (TB) therapeutics have stirred renewed interest in host-directed interventions, including the role of antibodies as adjunct therapeutic agents. This study assessed the binding efficacy of two novel IgG1 opsonic monoclonal antibodies (MABs; GG9 & JG7) at 5, 10, and 25 µg/mL to live cultures of Mycobacterium tuberculosis, M. avium, M. bovis, M. fortuitum, M. intracellulare, and M. smegmatis American Type Culture Collection laboratory reference strains, as well as clinical susceptible, multi-drug resistant, and extensively drug resistant M. tuberculosis strains using indirect enzyme-linked immunosorbent assays. These three MAB concentrations were selected from a range of concentrations used in previous optimization (binding and functional) assays. Both MABs bound to all mycobacterial species and sub-types tested, albeit to varying degrees. Statistically significant differences in MAB binding activity were observed when comparing the highest and lowest MAB concentrations (p < 0.05) for both MABs GG9 and JG7, irrespective of the M. tuberculosis resistance profile. Binding affinity increased with an increase in MAB concentration, and optimal binding was observed at 25 µg/mL. JG7 showed better binding activity than GG9. Both MABs also bound to five MOTT species, albeit at varied levels. This non-selective binding to different mycobacterial species suggests a potential role for GG9 and JG7 as adjunctive agents in anti-TB chemotherapy with the aim to enhance bacterial killing.

Risk analysis and seroprevalence of bovine ephemeral fever virus in Punjab, Pakistan.

Bovine ephemeral fever (BEF) is a vector-borne viral disease caused by the RNA virus which belongs to the genus Ephemerovirus and the family Rhabdoviridae. To evaluate the effect of the risk factors like the breed of cattle and buffaloes, age, sex, lactation, housing and region on the bovine ephemeral fever virus (BEFV) prevalence, ELISA and virus neutralisation (VN) tests (n = 600) were performed for the BEFV prevalence. The seroprevalence in cattle was 45.6% and 42% by ELISA and VN, respectively (P = 0.001). The breed-wise seropositive ratio was (55-64%) in cattle and (22.5-18.3%) in buffaloes by VN and ELISA. The sex-wise prevalence was (40-49.4%) in females and (35.8-46%) in males by VN and ELISA in cattle and a similar prevalence was reported in buffaloes. The age-wise prevalence in bovines by ELISA was 5.33, 22.66 and 17.66% in the age group < 1 year, 1-3 years and > 3 years, respectively. The disease prevalence was higher in the age group of 1-3 years. The prevalence was higher during the 3rd lactation in bovines. The region-wise prevalence was higher in the 07 districts while lower (18-21%) in Rawalpindi District by VN and ELISA, respectively (P = 0.001). Commercial dairy farms of cattle showed a higher disease prevalence (52% and 44%) than non-commercial farms (38% and 36%) by ELISA and VN, respectively (P = 0.227). Exotic cows showed higher disease prevalence (76.67% and 70%) by ELISA and VN. The mortality in bovines was 5% (7.7% and 2.3%) in the cattle and buffaloes. The case fatality of BEFV in bovines was 12.25%. There was a significant effect of the risk factors like the breed, age, sex, lactation, housing and region on the BEFV prevalence. This is the first comprehensive study of BEFV in Pakistan.

Anti-sperm Antibody Positivity in Men with Varicocele: A Systematic Review and Meta-Analysis.

PURPOSE: Despite the significant role of varicocele in the pathogenesis of male infertility, its association with anti-sperm antibodies (ASA) remains controversial. This systematic review and meta-analysis (SRMA) aims to investigate the frequency of ASA positivity in men with varicocele. MATERIALS AND METHODS: This SRMA is conducted in accordance with the Meta-analysis of Observational Studies in Epidemiology guidelines. We investigated the frequency of ASA positivity in ejaculates or serum of men with varicocele as compared to men without varicocele (controls). A literature search was performed using the Scopus and PubMed databases following the Population Exposure Comparison Outcome, Study Design model. Data extracted from eligible studies were meta-analyzed and expressed as odds ratios (ORs) and confidence intervals (CIs). RESULTS: Out of 151 abstracts identified during the initial screening, 6 articles met the inclusion criteria and were included in the meta-analysis. Using mixed antiglobulin reaction (MAR) assay, 61 out of the 153 (39.8%) patients with varicocele tested positive for ASA in their ejaculates as compared to 22 out of the 129 control subjects (17%, OR=4.34 [95% CI: 1.09-17.28]; p=0.04). Using direct or indirect immunobead test, 30 out of 60 cases diagnosed with varicocele (50%) had shown ASA positivity in their ejaculates as compared to 16 out of 104 controls (15.4%, OR=3.57 [95% CI: 0.81-15.68]; p=0.09). Using enzyme-linked immunosorbent assay (ELISA), out of 89 varicocele patients, 33 (37.1%) tested positive for serum ASA as compared to 9 out of 57 participants in the control group (15.8%, OR=7.87 [95% CI: 2.39-25.89]; p<0.01). CONCLUSIONS: This SRMA indicates that ASA positivity is significantly higher among men with varicocele when tested by direct method (MAR) or indirect method (ELISA). This data suggests an immunological pathology in infertile men with varicocele and may have implications for the management of these patients.

Clinical Value of Different Test Methods in Diagnosing Mycoplasma pneumoniae Infection in Children.

This research aimed to investigate the diagnostic value of passive particle agglutination test, Mycoplasma pneumoniae (MP) culture, cold agglutination test (CAT), enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction-capillary electrophoresis fragment analysis (PCR-CEFA) for MP infection. Children with respiratory tract infections suspected to be MP infection were subjected to passive particle agglutination test, MP culture, CAT, ELISA, and PCR-CEFA. A total of 146 children (81 males, 65 females, mean age: 5.74 ± 3.32 years, and mean course of disease: 9.07 ± 5.18 days) met the inclusion criteria. The positivity rate of MP detection by MP culture was 69.18% (101/146). Using the MP culture method as the standard, higher sensitivity and positive predictive value were found in the PCR-CEFA compared with the other 3 methods. Appropriate methods are selected following the advantages and disadvantages of pathogen detection, and pediatric MP infection is analyzed by integrating various test results.

Detection of intrathecal IgG antibody for varicella and measles diagnosis by evaluation and comparison of a commercial IgG chemiluminescent immunoassay with two ELISAs.

OBJECTIVES: Analyse alternative methods of intrathecal antibody detection by comparing chemiluminescent immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) techniques to determine if CLIA can replace ELISA in the diagnosis of CNS infections. METHODS: A panel of 280 paired samples-cerebrospinal fluid (CSF) and serum-with known antibody reactivities (Varicella, n = 60; Measles, n = 120) and negative samples (n = 100) were used to evaluate the performance of six serological test kits (Enzygnost, VirClia®, and Serion ELISA (Measles and Variella). RESULTS: For Measles virus IgG, the VirClia® IgG monotest revealed 97% and 94% positive and negative agreement to the Enzygnost as reference test, respectively. In contrast, Serion ELISA kits yielded values of 18% and 90%. For the Varicella Zoster virus (VZV) IgG, the VirClia® IgG monotest showed 97% and 90% positive and negative agreement compared to Enzygnost. The Serion ELISA kits showed values of 55% and 86%, respectively. ROC analysis revealed that the areas under the curve for Measles and VZV IgGs were 0.7 and 0.852, respectively, using the Serion kit, and 0.963 and 0.955, for Vircell S.L CLIA technique. VirClia® monotest values were calculated using an antibody index cut-off of 1.3. CONCLUSION: The findings indicate that CLIA testing can improve antibody detection in CSF samples, aiding the diagnosis of infectious neurological impairments.

Synthesis of salcaprozate sodium and its significance in enhancing pancreatic kininogenase absorption performance.

We conducted pharmacokinetic research wherein salcaprozate sodium (SNAC) was utilized as a penetration enhancer by incorporating it into pancreatic kininogenase (PK) to improve the bioavailability of pancreatic kininogenase enteric-coated tablets. We conducted in vitro studies on PK using the Caco-2 cell model and quantified PK levels using the enzyme-linked immunosorbent assay (ELISA) method. We conducted methodological verification by blending SNAC and PK powders into enteric-coated capsules, and studied the pharmacokinetic characteristics. Based on the PK transport assay, the cumulative permeation rates of the test group that employed a SNAC to PK ratio of 32:1, 16:1, 8:1, 4:1, and 2:1 were 13.574%, 7.597%, 10.653%, 3.755%, and 2.523%, respectively. We conducted a uniformity test on the powder that contained a blend of SNAC and PK. The relative standard deviations (RSDs) for both the power containing SNAC and the power not containing SNAC were less than 10%. Based on the methodological verification, in vivo pharmacokinetic study of PK met the experimental requirements. As indicated by the results of in vivo pharmacokinetic research on rats, the test group (This group used SNAC) had a PK AUC0-12 h of 5679.747 ng/L*h and t1/2 of 4.569 h, while the control group (This group did not use SNAC) had a PK AUC0-12 h of 4639.665 ng/L*h and t1/2 of 3.13 h. This study has established a low-cost, environmentally friendly, and safe SNAC synthesis route with high process yield suitable for industrial production. SNAC demonstrates an absorption-enhancing effect on PK, and the optimal ratio of SNAC to PK is determined to be 32:1.

A comparison of chemiluminescent immunoassay and enzyme-linked immunosorbent assay for detecting phospholipase A2 receptor antibody in primary membranous nephropathy.

Objective: The accurate detection of phospholipase A2 receptor (PLA2R) autoantibody is crucial in the diagnosis and monitoring of primary membranous nephropathy (pMN). While enzyme-linked immunosorbent assay (ELISA) is the commonly used detection method, its complexity and time-consuming nature pose challenges, especially for small sample sizes. Chemiluminescence immunoassay (CLIA) has emerged as a rapid alternative for clinical immunoassays. This study aims to compare the sensitivity, specificity, and precision of CLIA and ELISA in detecting PLA2R autoantibody. Method: A total of 145 patients with biopsy-confirmed primary membranous nephropathy and 85 patients with non-membranous nephropathy were enrolled in this comparative study. CLIA and ELISA were employed to test all samples for the presence of PLA2R autoantibodies. Statistical analysis of sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) was performed using SPSS 26.0. The diagnostic value of ELISA and CLIA for pMN was analyzed using the ROC curve, and Correlation analysis was performed using Spearman. Results: Serum levels of anti-PLA2R antibody in pMN group were significantly higher than those in nMN group(P < 0.05). The accuracy of CLIA for detecting anti-PLA2R antibody was 76.96%, while ELISA showed an accuracy of 74.78%. The sensitivity for CLIA was 64.83%, compared to 60% for ELISA. However, no statistically significant difference was observed between the two methods (P > 0.05). The overall qualitative agreement of anti-PLA2R detection was 93.35% (95% confidence interval[CI] 89.47-96.3). ROC curve analysis showed that AUC of anti-PLA2R antibody detected by ELISA and CLIA were 0.8737 (95% confidence interval [CI] 0.8270-0.9204), 0.8914 (95% confidence interval [CI]0.8495-0.9332), respectively. The Spearman correlation analysis revealed a significant correlation between them(P < 0.05). Notably, CLIA demonstrated a significant time-saving advantage, particularly when the sample size was less than 200, and especially when it was less than 20. Conclusion: CLIA and ELISA showed similar accuracy and consistency in detecting anti-PLA2R antibody for primary membranous nephropathy. However, CLIA exhibited a significant advantage in terms of automation and time-saving compared to ELISA, particularly for smaller sample sizes. This finding suggests that CLIA has the potential to become a preferred and widely adopted test in the future.

Establishment and evaluation of detection methods for process-specific residual host cell protein and residual host cell DNA in biological preparation.

To establish accurate detection methods of process-specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific-process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process-specific enzyme-linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non-process-specific commercial ELISA kit and the process-specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process-specific HCPs, could not be detected by the non-process-specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process-specific ELISA has high sensitivity in detecting process-specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future.

Epitope Mapping of an Anti-CD44v4 Monoclonal Antibody (C44Mab-108) Using Enzyme-Linked Immunosorbent Assay.

CD44 is a type I transmembrane glycoprotein and possesses various isoforms which are largely classified into CD44 standard (CD44s) and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 monoclonal antibody, C44Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C44Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10) and found that C44Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C44Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C44Mab-108 includes Asp280 and Trp281 of CD44v3-10.

Variability of BMP-2 content in DBM products derived from different long bone.

Demineralized bone matrix (DBM) has been regarded as an ideal bone substitute as a native carrier of bone morphogenetic proteins (BMPs) and other growth factors. However, the osteoinductive properties diverse in different DBM products. We speculate that the harvest origin further contributing to variability of BMPs contents in DBM products besides the process technology. In the study, the cortical bone of femur, tibia, humerus, and ulna from a signal donor were prepared and followed demineralizd into DBM products. Proteins in bone martix were extracted using guanidine-HCl and collagenase, respectively, and BMP-2 content was detected by sandwich enzyme-linked immunosorbent assay (ELISA). Variability of BMP-2 content was found in 4 different DBM products. By guanidine-HCl extraction, the average concentration in DBMs harvested from ulna, humerus, tibia, and femur were 0.613 ± 0.053, 0.848 ± 0.051, 3.293 ± 0.268, and 21.763 ± 0.344, respectively (p < 0.05), while using collagenase, the levels were 0.089 ± 0.004, 0.097 ± 0.004, 0.330 ± 0.012, and 1.562 ± 0.008, respectively (p < 0.05). In general, the content of BMP-2 in long bones of Lower limb was higher than that in long bones of upper limb, and GuHCl had remarkably superior extracted efficiency for BMP-2 compared to collagenase. The results suggest that the origin of cortical bones harvested to fabricate DBM products contribute to the variability of native BMP-2 content, while the protein extracted method only changes the measured values of BMP-2.

Preparation and Immunochemical Characterization of a Water-Soluble Gluten Peptide Fraction for Improving the Diagnosis of Celiac Disease.

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood.

Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

Comparative evaluation of rapid plasma reagin and ELISA with Treponema pallidum hemagglutination assay for the detection of syphilis in blood donors: a single center experience.

INTRODUCTION: The prime responsibility of blood transfusion services in India is to provide safe blood. The donated blood is tested for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), malaria and syphilis. In India, the screening of donated blood for syphilis is performed by rapid plasma reagin (RPR) or venereal disease research laboratory (VDRL), whereas the World Health Organization (WHO) recommends screening of syphilis in blood donors by enzyme-linked immunosorbent assay (ELISA). Therefore, the aim of this study was to evaluate the performance of RPR and ELISA with the Treponema pallidum hemagglutination assay (TPHA - the gold standard) for the detection of syphilis in blood donors. METHODS: In this cross-sectional study, 1524 consecutive whole blood donors were screened from April to October 2022. All blood samples collected during the study period were tested by RPR, ELISA and the TPHA and the results obtained were compared. RESULTS: The seroprevalence of syphilis in blood donors in this study was 0.06% by RPR and 0.72% by ELISA and TPHA. On considering ELISA and the TPHA as the gold standard, ELISA had comparable sensitivity (100%), a higher specificity (100% vs. 99.34%), a higher positive predictive value (PPV - 100% vs. 9.1%) and no biological false positive/false negative results (0 vs. 10 false negatives) when compared to RPR. CONCLUSION: ELISA performed better as a screening assay than RPR in the detection of syphilis in blood donors, which is in agreement with the WHO recommendations for syphilis testing in blood donors with low prevalence.

A comparative study of NS1 and IgM using enzyme-linked immunosorbent assay and immunochromatography test for detection of dengue.

Background: Dengue is the most common arboviral infection that spreads by Aedes aegypti and Aedes albopictus mosquitoes, and is quickly gaining prominence as a major mosquito-borne viral disease. One of the major public health issues is dengue fever (DF), which can also cause dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Therefore, this study focused on comparison of dengue antigen non-structural protein (NS1) and immunoglobulin M (IgM) using enzyme-linked immunosorbent assay (ELISA) and immunochromatography test (ICT) for detection of dengue. Method: In a Tertiary Care Hospital (TCH), sociodemographic status of probable dengue cases from February 2021 to February 2022 was studied. The results of the Dengue Antigen NS1 and IgM ICT, Dengue NS1 Microlisa, and Dengue IgM Microlisa were compared in order to determine the effective one at managing patients and preventing complications like DHF and DSS. Results: In distribution of 100 ICT reactive samples, 50% were NS1 reactive and 50% were IgM reactive. One hundred ICT reactive samples were further processed for IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for both NS1 and IgM. Sensitivity and specificity of NS1 ICT were 89.3% and 71.4% and that of IgM ICT was 88% and 64.5%. As a result of antigenic cross-reactivity, false positive cases were reported. Platelet count of the patients was correlated with an optical density (OD) value of ELISA for both NS1 and IgM. In the present study, patients having low platelet count showed high OD value. Conclusion: In cases of severe thrombocytopenia (platelet count <50000), early diagnosis by screening ICT and confirmation by ELISA (NS1 and IgM) would reduce the complications like DHF and DSS.

Relationship of Serum Prestin Levels to the Severity of Sensorineural Hearing Loss.

OBJECTIVE: Prestin is an outer hair cell (OHC) protein responsible for increasing cochlear sensitivity and has been proposed as a biomarker. We aimed to evaluate whether the serum prestin level is related to the severity of chronic sensorineural hearing loss (SNHL). METHODS: Ninety subjects were recruited from the patient base at Samarra public hospitals and clinics in Iraq from January to October of 2022. They were divided into three groups equally: a group of healthy people without hearing loss (G0), a group with moderate SNHL (G1), and a group with severe SNHL (G2). The subjects ranged from 20 to 80 years of age and included 51 males and 39 females. Blood samples were collected, then serum was separated, and enzyme-linked immunosorbent assays were performed to quantify the levels of prestin. RESULTS: Hearing thresholds were sequentially statistically higher across the three groups. While prestin levels were significantly higher in G1 and G2 than that in G0, there were no differences between the G1 and G2 levels. Serum prestin levels were positively correlated with hearing thresholds in G1, but not G2. CONCLUSION: Our results suggest that in the clinical setting, prestin is sensitive to chronic mild to moderate SNHL (i.e., up to 40-60 dB), not more severe loss. This range is consistent with the added sensitivity provided by OHCs in the cochlea and provides support for prestin as a biomarker of OHC-mediated SNHL.

Evaluation of the ability of commercial enzyme-linked immunosorbent assays to measure mouse tissue factor.

Background: Tissue factor (TF) is the primary cellular initiator of the blood coagulation cascade. Increased levels of TF expression on circulating monocytes or on extracellular vesicles (EVs) are associated with thrombosis in a variety of diseases, including sepsis and COVID-19. Objectives: Here, we aimed to evaluate the ability of 4 commercial TF enzyme-linked immunosorbent assays (ELISAs) to measure mouse TF in cells and plasma. Methods: We used 4 commercial mouse TF ELISAs (SimpleStep, R&D Systems, MyBioSource [sandwich], and MyBioSource [competitive]). We used recombinant mouse TF (rmTF; 16-1000 pg/mL), cell lysates from a TF-expressing mouse pancreatic cancer cell line, and plasma and EVs isolated from plasma from mice injected with vehicle or bacterial lipopolysaccharide (LPS). Results: The 2 MyBioSource kits failed to detect rmTF or TF in cell lysates. The SimpleStep and R&D kits detected rmTF in buffer or spiked into plasma in a concentration-dependent manner. These kits also detected TF in cell lysates from a mouse pancreatic cancer cell line. A higher signal was observed with the SimpleStep kit compared to the R&D kit. However, the SimpleStep and R&D kits failed to detect TF in plasma or EVs from LPS-treated mice. Conclusion: Our results indicate that some commercial ELISAs can be used to measure mouse TF levels in cell lysates but they cannot detect TF in plasma or EVs from endotoxemic mice.

Can Erythropoietin Open a Novel Avenue for Periodontal Regeneration?

INTRODUCTION: Periodontitis is a dramatic inflammatory disease, representing vigorous interactions between specific causative pathogens and host immune responses resulting in the activation of the destructive inflammatory cascade with the subsequent irreversible destruction of the teeth-supporting apparatus. AIM: This study aims to evaluate the effect of using erythropoietin (EPO) injectable hydrogel, as an additional therapeutic option to scaling and root planing (SRP) in the treatment of stage II periodontitis patients, and to assess its effect on the level of osteocalcin and interleukin (IL)-1ß in the gingival crevicular fluid (GCF). METHODOLOGY: A total number of 40 patients clinically diagnosed with stage II periodontitis were included. The participants were allocated into two equal groups: study and control groups. Patients in the control group received SRP, while those in the study group received SRP followed by injectable hydrogel containing EPO. Clinical parameters such as plaque index (PI), gingival index (GI), probing pocket depth (PPD), and clinical attachment level (CAL) were assessed at baseline and two months post treatment. GCF samples were collected at baseline and two months post treatment from both groups to analyze GCF IL-1ß and osteocalcin levels using enzyme-linked immunosorbent assay (ELISA). RESULTS: Significant reductions in all tested clinical parameters were revealed in both groups in comparison to baseline values. A marked significant reduction in GCF IL-1ß level was detected in the study group. However, two months post treatment, the osteocalcin level was decreased significantly in both groups. CONCLUSION: This preliminary study shows great promise for the local application of EPO hydrogel as an adjunct to SRP for the management of stage II periodontitis.