Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

Introduction: Canine leishmaniosis (CanL) is a systemic disease that affects dogs. When multiplication of the parasite cannot be controlled, dogs consistently show high levels of antigen and IgG antibodies, which lead to the formation of circulating immune complexes (CIC). Timely intervention to reduce the parasite load and CIC levels is crucial for preventing irreversible organ damage. However, a diagnostic test to quantify CIC levels is currently lacking. Methods: In this real-world study, we aimed to examine the performance of a new ELISA to measure CIC levels in dogs naturally infected with Leishmania infantum. Thirty-four dogs were treated according to their clinical condition and followed for 360 days. Before (day 0) and after treatment (days 30, 90, 180, 270, and 360), all dogs underwent a physical examination, and blood samples were obtained for CBC, biochemical profile, serum protein electrophoresis and IFAT. Serum PEG-precipitated CIC were determined by ELISA. Results: Our results indicate higher CIC levels in dogs in advanced disease stages showing higher antibody titres (p < 0.0001, r = 0.735), anemia (p < 0.0001), dysproteinemia (p < 0.0001), and proteinuria (p = 0.004). Importantly, dogs responding well to treatment exhibited declining CIC levels (p < 0.0001), while in poor responders and those experiencing relapses, CIC were consistently elevated. CIC emerged as a robust discriminator of relapse, with an area under the curve (AUC) of 0.808. The optimal cut-off to accurately identify relapse was an optical density of 1.539. Discussion: Our findings suggest that declining CIC levels should be expected in dogs showing a favorable treatment response. Conversely, in dogs displaying a poor response and recurrent clinical relapses, CIC levels will be high, emphasizing the need for vigilant monitoring. These findings suggest that CIC could serve as a valuable biomarker for disease progression, treatment efficacy, and relapse detection in CanL. Our study contributes to enhancing diagnostic approaches for CanL and underscores the potential of CIC as a complementary tool in veterinary practice. As we move forward, larger studies will be essential to confirm these findings and establish definitive cut-offs for clinical application.

Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena.

BACKGROUND: Antigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA. METHODS: We report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland-Altman (BA) analysis and Lin's concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA. RESULTS: The TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin's CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin's CCC: 0.24) but increased on days 28 and 90 PI (Lins' CCC: 0.88 and 0.98, P < 0.001). CONCLUSIONS/SIGNIFICANCE: The TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.

Epidemiological assessment of hepatitis E virus infection among 1565 pregnant women in Siem Reap, Cambodia using an in-house double antigen sandwich ELISA.

AIM: This study investigated hepatitis E virus (HEV) prevalence among pregnant women in Siem Reap, Cambodia, by developing a cost-effective, user-friendly in-house enzyme-linked immunosorbent assay (ELISA) for detecting total anti-HEV immunoglobulins (Ig). METHODS: The in-house ELISA was designed for large-scale screening in resource-limited settings. Its performance was benchmarked against two commercial tests: the Anti-HEV IgG EIA (Institute of Immunology, Co. Ltd) and the Anti-HEV IgG RecomLine LIA (Mikrogen). The in-house ELISA demonstrated a sensitivity of 76% and 71.4%, and a specificity of 94.1% and 98.6%, against the two commercial tests, respectively, with overall agreement rates of 92.4% and 94.3%. RESULTS: Among 1565 tested pregnant women, 11.6% were anti-HEV positive. Prevalence increased with age, particularly in women aged 35-40 years and over 40 years. No significant associations were found with education, number of children, family size, or history of blood transfusion and surgery, except for the occupation of the family head as a public officer. Of the total anti-HEV positive women, 22.7% had anti-HEV IgM, indicating recent or ongoing infection. CONCLUSION: The study concluded that the in-house ELISA is a viable option for HEV screening in regions with limited resources due to its high accuracy and cost-effectiveness. It is particularly suitable for large-scale studies and public health interventions in areas where HEV is endemic and poses a significant risk to pregnant women.

Highly selective and sensitive immunoassays for flurogestone acetate analysis in goat milk: From rational hapten design and antibody production to assay development.

The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC50 values from 0.17 to 0.45 µg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 µg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples.

Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis.

BACKGROUND: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. METHODS: Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. RESULTS: The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. CONCLUSION: The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

Comparison of two serological diagnosis tests for bovine paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (PTBC), a chronic infectious granulomatous enteritis of ruminants. The PTBC diagnosis with commercial ELISA has limitations in sensitivity and specificity, and its results depend on the state of progress of the disease. This research aimed to evaluate two different ELISAs: (a) an "in-house" ELISA with a sonicated antigen obtained from a MAP I47 strain, and (b) a commercial ELISA. In total, the evaluated sample consisted of 394 bovine serum samples from 12 farms in Argentina with high (5-9%) and low (≤ 0.05%) prevalence of PTBC. The evaluation of the new antigen (2.5 µg/mL) was against a 1:50 dilution of the M. phlei faced sera. The cut-off point, sensitivity, and specificity determinations of both techniques were by ROC curve analysis. The area under the curve for the I47 ELISA was 0.9 (CI 95%, 0.93-0.97). With a cut-off point of 8.8%, the sensitivity was 84.3% and the specificity 96.6%. The agreement between both techniques was 0.7 (CI 95%, 0.6-0.8). These results indicate a high discriminative capacity to differentiate positive and negative bovine sera of MAP infection with the I47 ELISA. This result would represent an advantage to dispense with the imported kit.

Inter-laboratory comparison of routine autoantibody detection methods for autoimmune neuropathies and myasthenia gravis.

Serological tests are important to detect autoantibodies (autoAbs) in patients with autoimmune neuropathies (AN) and myasthenia gravis (MG) as they are biomarkers for diagnosis, stratification, treatment selection, and monitoring. However, tests to detect autoAbs frequently lack proper standardization and results differ across diagnostic laboratories. We compared results for tests routinely performed in Spanish diagnostic laboratories to detect AN and MG autoAbs. In the Spanish Society of Immunology Autoimmunity Group national workshop, serum samples from 13 patients with AN or MG were tested for anti-ganglioside, anti-myelin-associated glycoprotein (MAG), anti-nicotinic acetylcholine receptor (AChR), and anti-muscle-specific kinase (MuSK) autoAbs using reference methods and were distributed for analysis to 27 participating laboratories using their routine methods. Overserved were inter-laboratory variability and worryingly low sensitivity, especially for anti-ganglioside immunoglobulin G and anti-MAG autoAb detection. This pilot study reflects autoAbs detection state of the art in AN and MG testing in leading diagnostic laboratories in Spain, highlighting the need for standardization prior to clinical use.

Evaluation of the Circulatory Levels of Heat Shock Protein 60 Levels in Periodontitis and Cardiovascular Disease Patients.

Introduction: HSP is arguably the most thoroughly studied self-antigens connected to Cardio Vascular Diseases (CVD) and periodontal disease. Hence, the major goal of this analysis was to determine the amount of HSP60 in patients' Chronic Periodontitis (CP) patients' serum. Materials and Methods: The current investigation involved 100 patients in all. Based on the patients' periodontal and cardiovascular health, the patients were divided. The patients were made aware that this research had no direct bearing on disease treatment or cure. Results: In contrast to periodontal disease, which had a mean serum HSP60 of 59.94 ng/dl, CVD had a mean serum HSP60 of 85.98 ng/dl. When compared to periodontal disease, the CVD increased significantly (P < 0.05, 0.03). Discussion and Conclusion: We emphasize the function of HSP60 in the pathophysiology of individuals with chronic periodontitis based on the findings of the current investigation. Serum HSP60 concentrations can serve as a biomarker for periodontal inflammation. More longitudinal and interventional research with a larger sample size is required to validate the present findings. In periodontal therapies, targeting HSP60 may enhance results.

Assessment of IL-8 Levels in Saliva of Healthy and Chronic Periodontitis Individuals.

Inflammation of the gums and other tissues supporting the teeth, as well as gradual loss of attachment and bone, are the results of chronic periodontitis, an infectious illness. During inflammation, a group of low molecular weight proteins called cytokines facilitate a complex interaction between inflammatory cells (such neutrophils) and other cellular components in connective tissue. The cytokine interleukin-8 (IL-8) is a potent neutrophil chemoattractant. Therefore, it is possible that IL-8 is crucial to the development of periodontitis's pathology. Objectives: 1) To estimate concentration of IL-8 levels in healthy individuals and chronic periodontitis individuals. 2) To compare IL-8 levels in healthy and chronic periodontitis individuals. Materials and Methods: Participants in this research will be recruited from among those who visit the outpatient department (OPD) at the NGH Institute of Dental Sciences and Research Centre, Belagavi, run by the Maratha Mandal. Control Group: Subjects with no clinical attachment loss (CAL) and a probing depth of 3.0 mm are considered to be periodontally healthy. Those in Group 2 (chronic periodontitis) have a chronic form of the disease, as shown by a probing pocket depth (PPD) of less than 5 mm and CAL of less than 2 mm. Unstimulated saliva sample will be collected in a 5 mL wide-mouthed sterile container by spitting method. Samples collected will be centrifuged. The supernatant is collected and stored at -80°C and then assayed for IL-8 concentration by using the standardized IL-8 ELISA kit.

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies.

African swine fever virus (ASFV) poses a significant threat to the global swine industry. Currently, there are no effective vaccines or treatments available to combat ASFV infection in pigs. The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig. Recently, a lower virulent ASFV isolate with a deleted EP402R gene (CD2v-deleted) has been reported in China, which further complicates the control of ASFV infection the pig farms. Furthermore, an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain. Therefore, it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections. In this study, two recombinant ASFV-p72 and -CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels. Subsequently, eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened. Following the production of these nanobodies with horse radish peroxidase (HRP) fusion proteins, the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs (cELISAs) to detect anti-ASFV antibodies. The two cELISAs exhibited high sensitivity, good specificity, repeatability, and stability. The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6% and 97.6%, respectively. Collectively, the two cELISA for detecting antibodies against ASFV demonstrated ease of operation, a low cost, and a simple production process. The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV, and they play an important role in monitoring ASFV infections in pig farms.

A candidate competitive ELISA based on monoclonal antibody 3A8 for diagnosis of contagious bovine pleuropneumonia.

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides (Mmm) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study's protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens. KEY POINTS: • A Mmm-specific diagnostic marker was obtained based on protein characteristics. • A cELISA was established for CBPP serum antibody detection. • The serological investigation was conducted for CBPP in the border areas of China.

Novel Piperazine Derivatives as Potent Antihistamine, Anti-Inflammatory, and Anticancer Agents, their Synthesis and Characterization.

INTRODUCTION: In this study, a series of novel piperazine derivatives were synthesised with high-to-good yields, and their structural analogies were confirmed using FTIR, 1H-NMR, and LC-MS techniques. METHOD: The synthesised compounds were evaluated for antioxidant and antimicrobial activities. Among the four synthesised piperazine derivatives, compound PD-2 exhibited relatively good antioxidant activity, with an IC50 value of 2.396 µg/mL, while the other three derivatives showed moderate to low antioxidant activity. Furthermore, compound PD-2 displayed antimicrobial activity against Pseudomonas aeruginosa, a gram-negative bacterium, and Candida albicans, a fungus. However, all four compounds showed strong resistance against grampositive bacteria, Staphylococcus aureus. RESULT: Additionally, compound PD-1 exhibited significant antihistamine activity, eliciting an 18.22% reduction in histamine levels. Both PD-1 and PD-2 demonstrated noteworthy anti-inflammatory activity in a dosedependent manner (5-10µM), leading to the inhibition of nitrite production up to 39.42% and 33.7% at higher concentrations (10 µM) and inhibition of tumour necrosis factor-alpha (TNF-α) generation up to 56.97% and 44.73% at 10 µM, respectively. Additionally, both novel molecules PD-1 and PD-2 effectively restrained the growth of HepG2 cells in a manner that is dependent on the dosage up to 55.44% and 90.45% at the highest concentrations (100 µg/mL), respectively. CONCLUSION: These findings substantiate the rationale for further investigation into the novel series of persuasive piperazine analogues as potential agents with anti-inflammatory, antihistamine and anticancer properties.

Nanozyme-assisted molecularly imprinted polymer-based indirect competitive ELISA for the detection of marine biotoxin.

Saxitoxin (STX), which is produced by certain dinoflagellate species, is a type of paralytic shellfish poisoning toxin that poses a serious threat to human health and the environment. Therefore, developing a technology for the convenient and cost-effective detection of STX is imperative. In this study, we developed an affinity peptide-imprinted polymer-based indirect competitive ELISA (ic-ELISA) without using enzyme-toxin conjugates. AuNP/Co3O4@Mg/Al cLDH was synthesized by calcining AuNP/ZIF-67@Mg/Al LDH, which was obtained by combining AuNPs, ZIF-67, and flower-like Mg/Al LDH. This synthesized nanozyme exhibited high catalytic activity (Km = 0.24 mM for TMB and 132.5 mM for H2O2). The affinity peptide-imprinted polymer (MIP) was imprinted with an STX-specific template peptide (STX MIP) on a multi-well microplate and then reacted with an STX-specific signal peptide (STX SP). The interaction between the STX SP and MIP was detected using a streptavidin-coated nanozyme (SA-AuNP/Co3O4@Mg/Al cLDH). The developed MIP-based ic-ELISA exhibited excellent selectivity and sensitivity, with a limit of detection of 3.17 ng/mL (equivalent: 0.317 µg/g). Furthermore, the system was validated using a commercial ELISA kit and mussel tissue samples, and it demonstrated a high STX recovery with a low coefficient of variation. These results imply that the developed ic-ELISA can be used to detect STX in real samples.

Modulation and Determination of the Status of Inflammasomes in Leishmania-Infected Macrophages.

Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1ß from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1ß result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.

Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 - 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

Differential cytokine expression in gastric tissues highlights helicobacter pylori's role in gastritis.

Helicobacter pylori (H. pylori), known for causing gastric inflammation, gastritis and gastric cancer, prompted our study to investigate the differential expression of cytokines in gastric tissues, which is crucial for understanding H. pylori infection and its potential progression to gastric cancer. Focusing on Il-1ß, IL-6, IL-8, IL-12, IL-18, and TNF-α, we analysed gene and protein levels to differentiate between H. pylori-infected and non-infected gastritis. We utilised real-time quantitative polymerase chain reaction (RT-qPCR) for gene quantification, immunohistochemical staining, and ELISA for protein measurement. Gastric samples from patients with gastritis were divided into three groups: (1) non-gastritis (N-group) group, (2) gastritis without H. pylori infection (G-group), and (3) gastritis with H. pylori infection (GH-group), each consisting of 8 samples. Our findings revealed a statistically significant variation in cytokine expression. Generally, cytokine levels were higher in gastritis, but in H. pylori-infected gastritis, IL-1ß, IL-6, and IL-8 levels were lower compared to H. pylori-independent gastritis, while IL-12, IL-18, and TNF-α levels were higher. This distinct cytokine expression pattern in H. pylori-infected gastritis underscores a unique inflammatory response, providing deeper insights into its pathogenesis.

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

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

Facile construction of sandwich ELISA based on double-nanobody for specific detection of α-hemolysin in food samples.

α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.

Trends in immunological markers of transfusion transmissible infections among blood donors in Mamfe District Hospital, Southwest Cameroon.

BACKGROUND: Blood transfusion is associated with exposure to blood Transfusion Transmissible Infection (TTIs). The threat posed by the blood-borne pathogens is disproportionately distributed in different healthcare facilities in Cameroon. Thus, there is a need for continuous surveillance of TTIs in the country. This study aimed to assess the screening procedure for blood transfusion and determine the trend in immunological markers of TTIs among blood donors at the Mamfe District Hospital. METHODS: A prospective descriptive, cross-sectional and analytical study was conducted at Mamfe District Hospital from March to May 2022. A total of 165 blood donors were recruited by the consecutive sampling method. Donors were screened using both Rapid diagnostic tests,T. pallidum haemagglutination test and indirect enzyme-linked immunosorbent assay (ELISA) for the detection of TTIs. Data generated was entered into an Excel spreadsheet and analysed using the statistical software R, version 4.2.0. Statistical analysis included descriptive statistics of percentages, means ± standard deviation, and student t-test was used to compare both diagnostic techniques, and was considered significant when p < 0.05. RESULTS: A hundred and sixty-five donors were enrolled in the study with a male preponderance giving a male-female sex ratio of 22.5 and a mean age of 32.23 ± 8.60 years. The majority (75.2%) of the donors were of the O-positive blood type, repeat donors (69.1%) and were mainly family replacement and paid donors as against the voluntary blood donors (39.4% and 37.0% vs. 23.6% respectively). overall TTIs prevalence was 18.78% (31/165) (), with HBsAg being the most predominant marker at 12.12% (20/165) followed by Treponema pallidum, HCV and HIV antibodies at 4.85 (8/165), 1.21%(2/165), 0.60% (1/165) respectively. Except for the HBV, The prevalence of TTIs was higher when using a single RDT than the ELISA test, and the difference was significant (p < 0.05). CONCLUSION: Bloodborne pathogens remain a major menace to safe blood transfusion practice in Mamfe district hospital and their detection could be easily missed if the RDT method alone is used for donor screening. Therefore, the donor screening protocol in Mamfe District Hospital should systematically incorporate a confirmation diagnostic test such as ELISA.