Rapid immunochemical methods for the analysis of proquinazid in strawberry QuEChERS extracts.

Proquinazid is a new-generation fungicide authorized in the EU for combating powdery mildew infections in high-value crops. Due to the perishable nature of fruits, alternative analytical methods are necessary to protect consumer's health from pesticide residues. Currently, immunoassays are a well-established approach for rapidly monitoring chemical contaminants. However, the production of high-quality immunoreagents, such as antibodies and bioconjugates, is essential. This study presents a newly designed hapten that maintains the characteristic moieties of proquinazid unmodified. The linear aliphatic substituents of this molecule were used to introduce the spacer arm. A three-step synthesis strategy was optimized to prepare a hapten that displays the entire 6-iodoquinazolin-4(3H)-one moiety with excellent yields. The N-hydroxysuccimidyl ester of the hapten was activated and purified to prepare a protein conjugate with high hapten density, which was used as an immunogen. Antibodies were raised and competitive enzyme-linked immunosorbent assays were developed. To enhance the assay's sensitivity, two additional heterologous haptens were prepared by modifying the halogenated substituent at C-6. The optimized assays demonstrated low limits of detection in buffer, approximately 0.05 µg/L. When applied to the analysis of proquinazid in QuEChERS extracts of strawberry samples, the immunoassays produced precise and accurate results, particularly in the 10-1000 µg/kg range.

A safe, cost-effective and high-throughput SARS-CoV-2 antigen capture ELISA suitable for large-scale screening in low-resource settings.

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195pg/100µL of N protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using ROC curve analysis, showing a diagnostic sensitivity of 67.78% and specificity of 100%. Sensitivity improved to 74.32% when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasyTM COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening.

Serological immune biomarker for disease severity in dengue-infected pediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using an enzyme-linked immunoassay assay system which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

Development and Bayesian validation of a competitive inhibition ELISA for detection of antibodies against Brucella abortus in cattle.

Bovine brucellosis is a zoonotic disease caused by Brucella abortus, responsible for abortions in cows. It is endemic in low- and middle-income countries, where the brucellosis control and eradication programs are based on compulsory vaccination, detection of infected cattle through serologic assays, and culling of infected animals at slaughterhouses. The development of high sensitivity and specificity, and low-cost serologic assays guarantee their implementation in countries where the disease is endemic. The aim of the present study was to develop and validate a competitive inhibition enzyme-linked immune assay (ciELISA) to detect anti-B. abortus antibodies in sera from cattle. The developed ciELISA was validated using 2833 serum samples from dairy and beef cattle. From these, 1515 sera were from uninfected cows that belonged to free of brucellosis herds and 1318 were from infected cows that belonged positive to brucellosis herds. Sera were analyzed with the developed ciELISA, the buffer plate antigen (BPA) test, and the complement fixation test (CFT). The brucellosis status of the herds was officially established according to the country legislation and consistent for at least 5 years and was defined for each cow using the CFT as gold standard. The cutoff for the ciELISA was calculated using a ROC curve and its sensitivity and specificity were analyzed using the Bayesian Latent Class Model (BLCM) approach. The agreement among tests was calculated using the kappa (κ) value. In addition, 15 calves were vaccinated with 3 × 1010 viable cells of B. abortus Strain 19 vaccine, and the dynamics of antibodies were measured by CFT, buffered plate antigen (BPA) test, and the developed ciELISA. The obtained cutoff for ciELISA was ≥ 47 percentage of inhibition (% I), at the BLCM approach the sensitivity was 99.01 % (95 % CI: 97.55-100) and the specificity 98.74 % (95 % CI: 97.68-99.8). The κ between the ciELISA and BPA was κ = 0.88 and between the ciELISA and CFT κ = 0.95. Antibodies against B. abortus were detected in all the vaccinated calves 7 days after vaccination (AV) by the three assays, at day 135 AV all the calves were negative to CFT (15/15), 93.3 % (14/15) to ciELISA and 73.3 % (11/15) to BPA, and at day 190 AV all the calves were negative to the three assays. The developed ciELISA showed a very good performance, could detect the majority of vaccinated animals as negative after 135 days and could be used for the detection of anti-B. abortus antibodies in serum samples for the brucellosis control and eradication program.

Techniques for improving the specificity of sandwich enzyme-linked immunosorbent assay-based drug screening.

ELISA assays are commonly used for drug screening by racing laboratories but are known to suffer from limited specificity. Inaccurate ELISA screening results are typically produced by non-specific antibody interactions or by the retention of chromogenic material in the sample well due to sample degradation. While confirmation of drug positives can be achieved by mass spectrometry, the follow-up of inaccurate ELISA screening results represents an unnecessary cost in staff time and reagents. This is particularly true in the case of rhEPO screening using sandwich ELISA assays, where the confirmation method requires up to 3 days to perform. While most racing laboratories purchase commercial ELISA kits, these products can be customised to provide increased specificity for enhanced screening of positive samples. The specificity of commercial sandwich ELISA kits can be improved by a variety of mechanisms including the addition of competing analyte specific antibodies, substitution of capture antibodies or by performing ELISA analysis with and without capture antibodies. Non-specific signals in difficult matrices such as canine urine can also be reduced by the addition of BSA solutions to the ELISA plate prior to the addition of samples.

Evaluation of commercial ELISA kits' diagnostic specificity for FAST diseases in wild animals.

Wild animals, sharing pathogens with domestic animals, play a crucial role in the epidemiology of infectious diseases. Sampling from wild animals poses significant challenges, yet it is vital for inclusion in disease surveillance and monitoring programmes. Often, mass surveillance involves serological screenings using enzyme-linked immunosorbent assay (ELISA) tests, typically validated only for domestic animals. This study assessed the diagnostic specificity of commercially available ELISA tests on 342 wild ruminant serum samples and 100 from wild boars. We evaluated three tests for foot-and-mouth disease: two for Peste des petits ruminants, two for Rift Valley fever and one for Capripox virus. Diagnostic specificity was calculated using the formula True Negative/(False Positive + True Negative). Cohen's kappa coefficient measured agreement between tests. Results showed high specificity and agreement across all tests. Specificity for foot-and-mouth disease (FMD) ranged from 93.89% for Prionics to 100% for IDEXX, with IDvet showing 99.6%. The highest agreement was between FMD IDvet and IDEXX at 97.1%. Rift Valley fever (RVF) tests, Ingezim and IDvet, achieved specificities of 100% and 98.83%, respectively. The optimal specificity was attained by retesting single reactors and inactivating the complement.Contribution: Commercially available ELISA kits are specific for foot-and-mouth disease and similar transboundary animal diseases and can be used for highly specific wild animal testing.

How does a Saccharomyces cerevisiae extract influence the components of isolated rotavirus particles from stool samples collected in a clinical setting from children?

Human Rotavirus (HRV) is the causative pathogen of severe acute enteric infections that cause mortality among children worldwide. This study focuses on developing a new and effective treatment for rotavirus infection using an extract from Saccharomyces cerevisiae, aiming to make this treatment easily accessible to everyone. 15 antigens and 26 antibodies were detected in serum and stool using ELISA. The titers of HRVq1, HRVq2, HRVC1, and HRVC2 on Vero cells were determined to be 1.2x106, 3.0x106, 4.2x106, and 7.5x105 (Plaque forming unit, PFU/ml) four days after infection, respectively. The HRVq1 isolate induced cytopathic effects, i.e., forming multinucleated, rounded, enlarged, and expanding gigantic cells. RT-PCR identified this isolate, and the accession number 2691714 was assigned to GeneBank. The molecular docking analysis revealed that nonstructural proteins (NSPs) NSP1, NSP2, NSP3, NSP4, NSP5, and NSP6 exhibited significant binding with RNA. NSP2 demonstrated the highest binding affinity and the lowest binding energy (-8.9 kcal/mol). This affinity was maintained via hydrophobic interactions and hydrogen bonds spanning in length from 1.12 Å to 3.11 Å. The ADMET and bioactivity predictions indicated that the yeast extract possessed ideal solubility, was nontoxic, and did not cause cancer. The inhibitory constant values predicted for the S. cerevisiae extract in the presence of HRV vital proteins varied from 5.32 to 7.45 mM, indicating its potential as a viable drug candidate. Saccharomyces cerevisiae extract could be utilized as a dietary supplement to combat HRV as an alternative dietary supplement.

Comparison of serological and molecular methods for differentiation between genotype A and genotype B strains of small ruminant lentiviruses.

Introduction: Small ruminant lentiviruses (SRLV) cause multisystemic, degenerative and chronic disease in sheep and goats. There are five genotypes (A, B, C, D and E), of which A and B are the most widespread. The purpose of this study was to evaluate the serotyping efficiency of the Eradikit SRLV Genotyping ELISA and the molecular typing efficiency of a newly developed nested real-time PCR targeting the long terminal repeat-gag (LTR-gag) region using samples from animals infected with subtypes of SRLV known to circulate in Poland. Material and Methods: A total of 97 sera samples taken from 34 sheep and 63 goats were immunoassayed, and 86 DNA samples from 31 sheep and 55 goats were tested with the PCR. All ruminants were infected with known SRLV strains of the A1, A5, A12, A13, A16, A17, A18, A23, A24, A27, B1 and B2 subtypes. Results: A total of 69 (80.2%, 95% confidence interval 71.6%-88.8%) out of 86 tested samples gave positive results in the PCR. In 17 out of the 86 (19.8%) samples, no proviral DNA of SRLV was detected. The differentiation between MVV (genotype A) and CAEV (genotype B) by PCR matched the predating phylogenetic analysis invariably. No cross-reactivity was observed. On the other hand, the proportion of samples genotyped the same by the older phylogenetic analysis and the Eradikit SRLV Genotyping ELISA was 42.3%. The test was unable to classify 40.2% of samples, and 17.5% of sera were incorrectly classified. Conclusion: Our results showed that the Eradikit SRLV genotyping kit is not a reliable method for predicting SRLV genotype, while the nested real-time PCR based on the LTR-gag region did prove to be, at least for genotypes A and B.

Resistin as a potential diagnostic biomarker for sepsis: insights from DIA and ELISA analyses.

PURPOSE: The primary objective of this investigation is to systematically screen and identify differentially expressed proteins (DEPs) within the plasma of individuals afflicted with sepsis. This endeavor employs both Data-Independent Acquisition (DIA) and enzyme-linked immunosorbent assay (ELISA) methodologies. The overarching goal is to furnish accessible and precise serum biomarkers conducive to the diagnostic discernment of sepsis. METHOD: The study encompasses 53 sepsis patients admitted to the Affiliated Hospital of Southwest Medical University between January 2019 and December 2020, alongside a control cohort consisting of 16 individuals devoid of sepsis pathology. Subsequently, a subset comprising 10 randomly selected subjects from the control group and 22 from the sepsis group undergoes quantitative proteomic analysis via DIA. The acquired data undergoes Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analyses, facilitating the construction of a Protein-Protein Interaction (PPI) network to discern potential markers. Validation of core proteins is then accomplished through ELISA. Comparative analysis between the normal and sepsis groups ensues, characterized by Receiver Operating Characteristic (ROC) curve construction to evaluate diagnostic efficacy. RESULT: A total of 187 DEPs were identified through bioinformatic methodologies. Examination reveals their predominant involvement in biological processes such as wound healing, coagulation, and blood coagulation. Functional pathway analysis further elucidates their engagement in the complement pathway and malaria. Resistin emerges as a candidate plasma biomarker, subsequently validated through ELISA. Notably, the protein exhibits significantly elevated levels in the serum of sepsis patients compared to the normal control group. ROC curve analysis underscores the robust diagnostic capacity of these biomarkers for sepsis. CONCLUSION: Data-Independent Acquisition (DIA) and Enzyme-Linked Immunosorbent Assay (ELISA) show increased Resistin levels in sepsis patients, suggesting diagnostic potential, warranting further research.

Efficiency of recombinant Ybgf in a double antigen-ELISA for the detection of Coxiella antibodies in ruminants.

Q fever is a zoonosis whose main reservoirs are domestic ruminants. Surveillance in these species is carried out mainly with serological tests, which, however, have limited diagnostic performance, and their manufacturing requires laboratories equipped with high biosafety requirements for antigen production. Recombinant ELISAs do not depend on these requirements and, being based on a single antigen, can reduce potential false positivity by identifying antibodies specific to a phase of infection. The aim of this study was to apply a new technology (dual antigen test) to a recombinant protein (Ybgf), an antigen produced in recombinant form and already used in previous studies for the design of an indirect ELISA. The successfully produced recombinant antigen was used to coat 96-well plates and, at the same time, another antigen aliquot was conjugated with HRP to obtain an HRP-conjugated Ybgf. After setting the test conditions, the results obtained with the recombinant double antigen test were compared with those obtained with a commercial assay (considered as reference assay) testing a total of 514 ruminant samples (280 goats and 234 cattle). A concordance of 86.2 and a Cohen's Kappa value of 0.72 were obtained, with no significant difference between the two species tested. Notably, the test proved to be highly specific, having correctly identified 250 out of 253 animals. This research represents an additional effort to use recombinant antigens to enhance serological methods in veterinary medicine. In a "one-health scenario", improving the performance of serological tests used in veterinary practice also means improving the surveillance of this infection.

Production and evaluation of anti-BP26 monoclonal antibodies for the serological detection of animal brucellosis.

Brucella BP26 proves to be a highly immunogenic antigen with excellent specificity in brucellosis detection. In China, the authorized use of the Bp26-deleted vaccine M5ΔBP26 for preventing small ruminant brucellosis highlights the importance of developing accurate detection methods targeting BP26, particularly for the diagnosis of differentiation between infected and vaccinated animals (DIVA). Using the traditional mouse hybridoma technique, we successfully obtained 12 monoclonal antibodies (mAbs) targeting BP26. The efficacy of these mAbs in detecting various animal brucellosis cases using the competitive ELISA method was evaluated. Among them, only the E10 mAb exhibited significant efficiency, being inhibited by 100, 97.62, and 100% of brucellosis-positive sera from cattle, small ruminants, and canines, respectively. The E10-based competitive enzyme-linked immunosorbent assay (cELISA) outperformed the BP26-based indirect enzyme-linked immunosorbent assay (iELISA) in accuracy, particularly for cattle and small ruminant brucellosis, with cELISA sensitivity reaching 97.62% compared to 64.29% for iELISA for small ruminants. Although cELISA showed slightly lower specificity than iELISA, it still maintained high accuracy in canine brucellosis detection. The epitope of mAb E10 was identified in the amino acid sequence QPIYVYPDDKNNLKEPTITGY, suggesting its potential as a diagnostic antigen for brucellosis. In conclusion, the E10-based cELISA presents an effective means of detecting animal brucellosis, particularly significant for DIVA diagnosis in China, where the BP26-mutant vaccine is widely used.

Inadequate Glucagon Suppression During OGTT in prediabetes: A Systematic Review and Meta-Analysis.

CONTEXT: Glucagon plays a role in the development of type 2 diabetes, yet its role in prediabetes (preDM) remains uncertain. OBJECTIVE: To evaluate glucagon levels in fasting state and its response to glucose inhibition in preDM through meta-analysis. METHODS: A systematic search across Pubmed, Embase, Web of Science, and Cochrane Library identified studies assessing glucagon levels during 75g oral glucose tolerance test (OGTT) in both preDM and normal glucose tolerance (NGT) cohorts. Data on glucagon, glucose and insulin were pooled using random-effect model. RESULTS: Although glucagon levels decreased in both preDM and NGT groups upon glucose challenge, glucagon levels at 0h, 0.5h, 1h and 1.5h in preDM were significantly higher compared to NGT, despite higher glucose levels at all time points and higher insulin levels at 0h, 1h, 1.5h and 2h during OGTT. Subgroup analysis revealed that in studies using the radioimmunoassay (RIA) method, glucagon levels in preDM were higher at 0.5h and 1h than NGT, while in studies using the Enzyme linked immunosorbent assay (ELISA) method, glucagon levels were similar to those of the NGT group despite higher glucose in preDM compared to NGT. Fasting glucagon level was inadequately suppressed in both impaired glucose tolerance (IGT) and impaired fasting glucose (IFG). Responsiveness to glucose inhibition was preserved in IFG, while glucagon level in IGT group at 0.5h after glucose intake was not suppressed and was higher than NGT. CONCLUSION: Glucagon was not adequately suppressed during OGTT in preDM. Glucagon dysregulation is a contributing mechanism underlying both IFG and IGT.

Epidemiological factors conditioning sarcoptic mange spreading in wild boar (Sus scrofa).

Sarcoptic mange is a widely distributed disease, with numerous potential hosts among domestic and wild animals. Nowadays it is considered a neglected re-emergent infection in humans. As a difference with domestic pigs, and even with several clinical cases reported in some European countries, it seems that Eurasian wild boars (Sus scrofa) have a low susceptibility to clinical mange. However, because of a case of confirmed transmission from Spanish ibex (Capra pyrenaica) to wild boar in the province of Tarragona, we planned a large-scale ELISA survey in the neighboring Valencian Community (SE Spain). We compared 419 wild boar sera from different management systems (fenced vs. open game estates), different ages (piglets, juveniles, and adults), with different behaviour (gregarious females of all ages and male piglets vs. solitary juveniles and adult males), from areas with different wild boar densities, different wild ruminant densities and different sarcoptic mange epidemiologic situations. The whole prevalence of antibodies against sarcoptic mange in the tested wild boars was 10.5%. No significant differences were found when comparing fenced and free ranging wild boars, males and females, gregarious vs. solitary individuals or among different ages. However, wild boar density was a relevant factor. In areas with a hunting bag of <1 wild boar/km2, considered as a low density of suids, the seroprevalence was 2.94%, but rose to 11.52% in high density districts, constituting a significant difference (p = 0.037). Low wild boar populations would act as a protective factor (OR 0.233; p = 0.049) against coming into contact with the mite. The wild ruminant densities or their sarcoptic mange status did not show any effect on wild boars seroprevalence against this disease. These results reinforce the suggested host-taxon Sarcoptes scabiei specificity and the independence of host-species foci.

A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

Quantification of DNA Methylation by ELISA in Epigenetic Studies in Plant Tissue Culture: A Theoretical-Practical Guide.

This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samples of commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA test to construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.

Evaluation of Somatic Antigens of Adult Toxocara helminthes for Detection of Human Toxocariasis.

We aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) to evaluate the presence of specific IgG against Toxocara canis and Toxocara cati somatic antigens on the serum of patients with toxocariasis. The sensitivity, specificity, positive and negative predictive values for indirect-ELISA were calculated by receiver operating characteristic curve (ROC) analysis and Youden's J using Likelihood ratio. All statistics were analysed and graphs are plotted using GraphPad Prism version 8.4.3 (Graph Pad Software, La Jolla, CA, USA), with 95% confidence interval (CI). The sensitivity, specificity, positive and negative predictive values for T. canis were 100%, 82%, 79% and 100%, respectively. The mentioned variables for T. cati were 97%, 82%, 78% and 98%, respectively. Five immune reactive bands of 38, 40, 72, 100 and 250 kDa were common in both species. Toxocara crude antigens were highly immunogenic in human sera. Immunoreactive bands against T. canis compared to T. cati somatic antigen were about two times more. Unlike Toxocara excretory-secretory antigen, that was homologue in two species, somatic antigens of T. canis and T. cati showed different immunoreactive bands in our western blot.

Serodiagnosis of nasal myasis in camels (Camelus dromedaries) in Egypt using third larval instar affinity-purified glycoprotein.

The larvae of Cephalopina titillator cause nasopharyngeal myiasis in camels, which parasitize the living tissues of the nasal and paranasal sinuses, pharynx, and larynx. C. titillator infestation adversely affects camel health, meat, and milk production, and can even cause death. In our study, to improve the immunodiagnosis of camel nasal myiasis, a sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed and evaluated using the Concanavalin-A (Con-A) affinity purification for the C. titillator-N-acetylglucosamine (Ct-GlucNAc) glycoprotein fraction from third larval instars as an antigen for detecting C. titillator antibodies. Crude antigens were prepared from larval instars of C. titillator and evaluated by indirect ELISA. The third C. titillator larval antigen (L3Ct) had the highest protein content (P < 0.001) and the best diagnostic value; chi-square = 235 (P < 0.001). Four glycoprotein fractions were purified separately from the L3Ct antigen by Con-A purification and evaluated. The Ct-GlucNAc glycoprotein fraction was the fraction of choice with the highest diagnostic accuracy (P < 0.05). Using Ct-GlucNAc as a coating antigen, indirect ELISA showed a 99.3% sensitivity for positive results in camel myiasis samples and 100% specificity for negative results in healthy camel samples. The diagnostic accuracy was 99.7%, and no cross reactivity was detected for other parasitic diseases. The indirect ELISA results were confirmed by the western immunoblotting which was characterized by comparing sera from naturally infested dromedary camels with C. titillator, sera from healthy camels and sera from camels with other parasitic infections (Echinococcus granulosus, Fasciola gigantica, Hard ticks; Hyalomma dromedarii, Trichostronglid sp., Eimeria spp., and Cryptosporidium sp.). Immunoreactive antigenic bands of 63, 50, 30 and 18 kDa were predominantly detected in sera from camels with nasopharyngeal myiasis and didn't react with healthy and camel's sera from other parasitic infections. However, seven immunoreactive bands appeared at 120, 70, 63, 48, 35, 29, and 19 kDa in the crude L3Ct antigen. In addition, a positive rate of C. titillator immunodiagnosis was detected by indirect ELISA (48.6%, chi-square = 483, P < 0.001), which was significantly greater than that of postmortem diagnosis (31%). In conclusion, the current study introduces a new diagnostic immunoaffinity glycoprotein fraction of C. titillator 3rd larval instar-based ELISA as a highly accurate, simple and fast method to detect specific antibodies of nasal myiasis in camels.

microRNA-106b-5p and Rab10: Potential Markers of Acute Myeloid Leukemia.

This study focuses on acute myeloid leukemia (AML), a condition with a 5-year survival rate below 30% despite various treatment options. Recent strides in targeted therapies have shown promise, leading to better outcomes with minimal toxicity. These advances underscore the importance of discovering new diagnostic and prognostic targets for AML. In this context, the authors investigated the expression of microRNA-106b-5p (miR-106b-5p), Rab10 mRNA, and Rab10 proteins in peripheral blood and bone marrow (BM) samples from both healthy individuals and AML patients at different stages of the disease (initial diagnosis, recurrence, and complete remission). This examination aimed to identify potential biomarkers for AML diagnosis, treatment, and prognosis. From June 2021 to December 2022, they collected 100 BM and peripheral blood samples. The relative expression of miR-106b-5p and Rab10 mRNA in the BM of AML patients was measured using Real-time polymerase chain reaction (qRT-PCR), while the relative expression of Rab10 protein in serum was determined using the ELISA method. The chromosomal karyotype of initially diagnosed patients was analyzed using the R tape. The qRT-PCR results revealed that the expression of miR-106b-5p and Rab10 mRNA were significantly higher in patients at initial diagnosis and recurrence compared with healthy individuals and those in complete remission (p < 0.001). They observed a significant reduction in the expression of miR-106b-5p, Rab10 mRNA, and Rab10 protein in the BM and peripheral blood of patients during complete remission (p < 0.05), as demonstrated by dynamic monitoring of five patients in the initial group. Furthermore, they found a close association between the expression of miR-106b-5p and the number of white blood cells at the initial diagnosis in AML patients (p < 0.05). Spearman correlation analysis revealed a positive correlation among miR-106b-5p, Rab10 mRNA, and Rab10 proteins (p < 0.05). The diagnostic potential of miR-106b-5p and Rab10 proteins was underscored by Receiver Operating Characteristic (ROC) curve analysis, which demonstrated their high accuracy in AML diagnosis (AUC: 0.944 and 0.853, respectively; p < 0.0001). Additionally, Kaplan-Meier survival analysis suggested that lower expression of these markers was associated with better prognoses (p < 0.05). In summary, their findings propose miR-106b-5p and Rab10 proteins as promising biomarkers for AML, offering insights for diagnosis, treatment, and prognosis.

Comparative assessment of the diagnostic performances of particle-based multianalyte technology and commercial ELISA for antiphospholipid autoantibody testing.

BACKGROUND: Diagnosing Antiphospholipid Syndrome (APS) relies heavily on laboratory findings, particularly the detection of specific antibodies like lupus anticoagulant (LA), IgG and/or IgM anti-cardiolipin (aCL), and IgG and/or IgM anti-ß2 glycoprotein 1 (aB2GP1). Although ELISA is widely used in the US for this purpose, standardization between different assay methodologies remains challenging, leading to significant variability across laboratories. Particle-based multi-analyte technology (PMAT) offers a streamlined one-step detection for all six antiphospholipid (aPL) autoantibodies, covering aCL and aB2GP1 of IgA, IgG, and IgM isotypes. METHODS: In this study involving 224 subjects, including 34 clinically diagnosed with APS, alongside 160 non-APS patients and 30 healthy donors, PMAT's performance was evaluated against commercial ELISA in detecting aPL antibodies. RESULTS: At the manufacturer's suggested cutoff, PMAT exhibited sensitivity comparable to ELISA, albeit with a low to moderate decrease in specificity for certain antibodies. With anti-CL IgM alone, PMAT displayed a 17.7% decrease in sensitivity, accompanied by a corresponding 31.1% increase in specificity compared to ELISA. However, applying a stricter cutoff (88-90% specificity), IgA and IgM antibodies yielded 5.9-17.6% higher sensitivities with PMAT, and IgG antibodies displayed similar sensitivity. CONCLUSIONS: In this study cohort, PMAT demonstrated higher or comparable sensitivity to that of commercial ELISA for all six aPL antibodies at a specificity cutoff near 90%. Notably, PMAT demonstrated superior sensitivity and specificity overall in detecting IgA aCL and aB2GP1 antibodies. This study highlights the potential of automated PMAT for detecting aPL antibodies in APS evaluation.

ELISA for leptospiral 3-hydroxyacyl-CoA dehydrogenase in urine is a promising screening tool for acute leptospirosis.

Introduction. Leptospirosis is a zoonotic disease that is prevalent worldwide. Leptospiral 3-hydroxyacyl-CoA dehydrogenase (3-HADH) is excreted in the urine of infected individuals. However, the potential use of 3-HADH as a biomarker for the diagnosis of leptospirosis using enzyme-linked immunosorbent assay (ELISA) has not been investigated. A technique that identifies Leptospira in a patient in urine sample will be valuable in regular diagnostics and epidemic scenarios, as opposed to existing serological approaches. This study aimed to develop and evaluate an ELISA that can detect 3-HADH in the urine of patients with confirmed acute leptospirosis and to assess its potential as a screening test for leptospirosis. Methods. Laboratory confirmation of acute leptospirosis was done by flaB-nested polymerase chain reaction (PCR) of plasma samples from suspected patients. ELISA-based determination of the presence of 3-HADH in the urine of PCR-positive patients versus PCR-negative patients matched for fever date was performed by coating ELISA plates with urine supernatants and using rabbit anti-3-HADH as the primary antibody. Receiver operating characteristic curve analysis was used to determine the cutoff values for the ELISA. The diagnostic measures between the PCR-positive and PCR-negative patients were compared using the Mann-Whitney U test. Results. In total, 158 febrile patients were assessed, of whom 121 (76.6 %) were male. Of the 15 flaB-nested PCR-positive patients, 12 were in the acute phase of the febrile illness. The best cutoff was an average optical density (ODav) value of 0.2200 for febrile patients. Sensitivity and specificity were 83.33% [95 % confidence interval (CI), 51.59-97.91 %) and 83.33 % (95 % CI, 76.05-89.13 %), respectively. The ODav values for PCR-positive patients in the acute phase of the disease (≤7 days of fever) were significantly higher than those for PCR-negative patients (P<0.001, U=114.0, z=-4.946). Conclusion. Detection of 3-HADH in urine by ELISA appears to be promising for the screening of acute leptospirosis in suspected patients.