Recently, two respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We developed and validated an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and choice of blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 μg/mL of DS-Cav1 and SC-TM, respectively, showed high coefficients of determination (r2 = 0.90). Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r2 = 0.90) and SC-TM (r2 = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM but not with the attachment protein.

Pretilachlor is a chloroacetamide herbicide, mainly used for the control of weeds and broadleaf weeds in rice, and widely used in China. For the detection of residues of pretilachlor in the en-vironment and food, a highly sensitive and specific monoclonal antibody against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47±2.35 μg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 μg/L was developed for the detection of pretilachlor residues in the environment and food crops. The specificity of the antibody was explained by computer simula-tions and experimental validation. No cross-reactivity of this monoclonal antibody to alachlor, acetochlor and metalaxyl, and it cross-reacted less than 3.0% to both butachlor and propisochlor. The limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 μg/L. The recoveries of all samples were 78.3%~91.3%. The ic-ELISA method was validated by high-performance liquid chromatography, thus, it can be used for residue detection of pretilachlor in the environment and grains.

(1) Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. (2) Methods: One hundred twelve 15-mer peptides covering the entire sequence of DTx protein were prepared by Spot-synthesis. Membrane-bound peptides immunoreactivity with sera from mice immunized with triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, MAPs synthesis, and ELISA development. (3) Results: Twenty epitopes were identified, being 2 in the signal peptide, 5 in the CD, 7 in the HBFT domain, and 5 in the RBD. Two 17-mer (CB/Tx-2/12 and CB/DTx-4-13) derived bi-epitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccined sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) either for huVS and miVS and when compared with a commercial ELISA test, demonstrate high performance. (4) Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated the specificity and eligibility to enter phase IIB studies of some epitopes to develop new and fast diagnostic assays.

Saliva is a promising matrix with several purposes. Our aim is to verify if salivary anti-SARS-CoV-2 antibody determination is suitable for monitoring immune response. One-hundred eighty-seven subjects were enrolled at University-Hospital Padova: 105 females (56.1%) and 82 males (43.9%), 95 (50.8%) children and 92 (49.2%) adults. Subjects self-collected saliva using Salivette; nineteen subjects collected three different samples within the day. A serum sample was obtained for all individuals. The N/S anti-SARS-CoV-2 salivary IgG (sal-IgG) and serum anti-SARS-CoV-2 S-RBD IgG (ser-IgG) were used for determining anti-SARS-CoV-2 antibodies. The mean (min-max) of age was 9.0 (1–18) for children and 42.5 (20–61) for adults. Of 187 samples, 63 were negative for sal-IgG (33.7%), while 7 were negative for ser-IgG (3.7%). Spearman’s correlation was 0.56 (p<0.001). Sal-IgG and ser-IgG levels were correlated with age, but not with gender, comorbidities, prolonged therapy, previous SARS-CoV-2 infection or time from last COVID-19 infection/vaccination. The repeatability ranged from 23.8% (7.4kAU/L) to 4.0% (3.77kAU/L). Linearity of the assay missed in 4/6 samples. No significant intra-subject differences were observed in sal-IgG across samples collected at different time points. Sal-IgG have a good agreement with ser-IgG. Non-invasive saliva collection represents an alternative method for antibody measurement, especially in children.

Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT responsible for protection remains to be clarified. Here, we have mapped the B-cell linear epitopes of the three chains of the CT protein (CTP) prepared by Spot synthesis. The immunoreactivity of sera from mice immunized with an oral, inactivated vaccine (Schankol†™) was measured against membrane-bound peptides for mapping. Results: Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three epitopes, TQTGFVRHDDGYVST (aa 66-77, Vc/TxA-3), KNGAIFQVE VPGSQN (aa 64-78, Vc/TxB-11), and LNDEHK (aa 90-95, Vc/TxP-16), were chosen to synthesize a multiple antigen peptide that was used to coat ELISA plates to screen immunized mouse sera as a test for an in vitro diagnostics for cholera. Conclusion: Vaccination with inactive CT-generated antibodies against multiple linear epitopes and the selected epitopes can be used to construct immunological reagents for rapid serological diagnosis of cholera with high sensitivity and specificity.

Myocardial infarction (MI) is a serious cardiovascular disease that can lead to death. Cardiac troponin I (cTnI) is a biomarker of MI in the circulation system. For detection cTnI protein, many method suffer the same problem, time-consuming. Main drawbacks are time consuming, have tedious operation, require an expensive reader for translating signals, and require non-accessible reagents. Our goal is to create a kit for cTnI protein testing, that the result can easily be read by color difference. The main breakthrough is, the resolution between different concentration can be told by eyes. The result will be future used to create a commercial, potable, convenient, daily-used rapid-test kit. In this study, a cTnI biosensor is proposed that can be read by the naked eye, performs diagnosis based on the pattern color, does not require a reader machine, is easy to operate, and is portable. Our work proposed a device that shortens the diagnosis time, has a quantitative analysis range of 0.32 –200 ng/mL in the human serum matrix, has a 0.32 ng/mL limit of detection (LOD), and shows many advantages comparing to traditional cTnI ELISA plate.

Neosporosis is an infection caused by the protozoan parasite Neospora caninum and has substantial veterinary hazards. Neosporosis cannot be controlled via vaccination or a chemotherapeutic agent. Thus, accurate diagnosis followed by isolation and culling of infected animals is regarded as the most efficient way of control. In vivo diagnosis often relies on serologic testing of the animals, and milk represents a non-invasive and easy to collect sample matrix. However, ELISAs specifically designed for the antibody detection in milk are sometimes not easily available and it is tempting to use ELISA kits designed for use in serum also for milk samples. Herein, we evaluated a widely used commercial ELISA (ID Screen® Neospora caninum competition Multispecies ELISA (ID. Vet, Grabels, France)), developed for detection of N. caninum antibodies in serum samples, for its performance with milk samples instead. Milk samples from dairy ruminants (cows, buffaloes, sheep, goats; n = 149) were tested in parallel with the serum ELISA and a commercial milk ELISA as a standard test (Neospora caninum Milk Competitive ELISA, ID. Vet, Grabels, France). The detected prevalence rates were 42 (28.2%), 26 (17.4%), and 26 (17.4%) using milk ELISA, serum ELISA, and both ELISAs, respectively. Sensitivity, specificity, positive predictive value, and negative predictive value for the serum ELISA used with milk samples were 61.9%, 100%, 100%, 87%, respectively: Agreement and Kappa value between the two ELISAs were 89.3%, and 0.70, respectively. High values of Pearson correlation coefficient (0.991) and area under ROC curve (0.789) demonstrated a high diagnostic performance of serum ELISA also in milk samples. Also, Bland-Altman plot and Histogram describing the frequency of distribution of ELISA optical densities confirmed the high agreement of both serum and milk ELISAs. The current results revealed a high specificity but moderate sensitivity of the serum ELISA used for milk samples compared with the milk ELISA. However, the excellent positive predictive value of the serum ELISA makes it an alternative option in case of unavailability of milk ELISA. With this study, we provided additional evidence that a widely used serum ELISA test kit may also be used for detection of N. caninum antibodies in milk samples.

Neurofilament light chain (Nf-L) is a well-known biomarker for axonal damage, however the corresponding circulating Nf-L analyte in CSF is poorly characterized. We, therefore, isolated new monoclonal antibodies against synthetic peptides, and these monoclonals were characterized for their specificity on brain-specific intermediate filament proteins. Two highly specific antibodies, ADx206 and ADx209, were analytically validated for CSF applications according to well-established criteria. Interestingly, using three different sources of purified Nf-L proteins, a significant impact on interpolated concentrations was observed. With a lower limit of analytical sensitivity of 100 pg/ml using bovine Nf-L as the calibrator, we were able to quantify the Nf-L analyte in each sample and these Nf-L concentrations were highly correlated to the Uman diagnostics assay (Spearman rho= 0.97, P<0.001). In the clinical diagnostic groups, the new Nf-L ELISA could discriminate patients with Alzheimer’s disease (AD, n=20) from frontotemporal lobe dementia (FTD, n=20) and control samples with subjective cognitive decline (SCD, n=20). Henceforth, this novel Nf-L ELISA with well-defined specificity and epitopes can be used to enhance our understanding of harmonizing the use of Nf-L as a clinically relevant marker for neurodegeneration in CSF.

Galectins play a role in lung cancer oncogenic pathways including apoptosis, t-cell immune responses, and tumor metastasis. Biomarkers that diagnose, prognose, and assist with treatment remain important, and galectins represent biomarkers that have the potential to impact lung cancer management through these pathways. Using enzyme-linked immunosorbent assay (ELISA) we examined galectin-1, -3, and -9 levels from lung cancer patient serum. Through comprehensive chart review: patient demographics, staging information, tumor biology, treatment data, and outcomes including overall survival (OS) and metastasis were retrospectively collected. Galectin levels were then compared to these numerous factors. Galectin-1, -3, and -9 levels did not correlate with cancer stage, however galectin-3 levels were lower in small cell lung cancer (SCLC) and squamous cell lung cancer than in adenocarcinoma. There was no correlation between galectin levels and previous treatment or future metastasis. Patients that had an abnormal galectin-1 level had decreased OS. Furthermore, patients with curative surgically resectable NSCLC with abnormal galectin-1 levels had decreased OS, but there was no difference in those with SCLC or that were non-resectable treated palliatively. These findings provide insight into galectin levels in various lung cancer histology and highlight the potential for galectin-1 to assist in prognosis of surgically resectable NSCLC.

Emerging evidence has shown that microRNAs play pivotal roles in wound healing. MicroRNA-21 (miR-21) was previously found to upregulate in order to fulfill an anti-inflammation role for wounds. Exosomal miRNAs have been identified and explored as essential markers for diagnostic medicine. However, the role of exosomal miR-21 in wounds has yet to be well studied. In order to facilitate the early management of poorly healing wounds, we developed an easy-to-use, rapid, paper-based microfluidic-exosomal miR-21 extraction device to determine wound prognosis in a timely manner. We isolated and then quantitatively examined exosomal miR-21 in wound fluids from normal tissues, acute and chronic wounds. Eight improving wounds displayed lower levels of exosomal miR-21 expression after wound debridement. However, four instances of increased exosomal miR-21 expression levels were notably associated with patients with poor healing wounds despite aggressive wound debridement, indicating a predictive role of tissue exosomal miR-21 for wound outcome. Paper-based nucleic acid extraction device provides a rapid and user-friendly approach for evaluating exosomal miR-21 in wound fluids as a means of monitoring wounds. Our data suggest that tissue exosomal miR-21 is a reliable marker for determining current wound status.

CC chemokine receptor 6 (CCR6) is one of the members of G protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, including B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. Coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, autoimmune diseases, and psoriasis. Therefore, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for patients. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), applicable for flow cytometry by immunizing a rat with N-terminal peptide of mCCR6. This study investigated the binding epitope of C6Mab-13 using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methods with the synthesized point mutated-peptides within 1-20 amino acids region of mCCR6. In ELISA, C6Mab-13 lost the reaction to the alanine-substituted peptide of D11A. The epitope of C6Mab-13 was identified to be Asp11 in ELISA. Furthermore, in SPR analysis, the dissociation constants (KD) could not be calculated for G9A and D11A mutants due to lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.

Tandem repeat proteins of 36 kDa (TRP36) are major immunoreactive proteins of Ehrlichia canis, which have been used in the serological diagnosis of different genotypes of the microorganism. The aim of this study was to evaluate the distribution of the American (USTRP36), Brazilian (BrTRP36) and Costa Rican (CRTRP36) genotypes of E. canis in Brazil, using ELISA assays. Serum samples of 815 dogs from 49 cities from all over Brazil were analyzed. Our results showed that 33.9% of the samples were reactive to the USTRP36 genotype and 32.6% to the BrTRP36 genotype. The two genotypes appeared to occur equally throughout Brazil, although the frequency of seropositivity was lower in the south than in the country’s other regions. Co-positivity for the American and Brazilian genotypes was also observed in 16% of samples. A few dogs (n=5; 0.6%) reactive to E. canis-TRP36 genotype (CRTRP36) were also detected in the northeast and southern regions. We conclude that the American and Brazilian genotypes of E. canis are distributed evenly in Brazil, especially in the tropical region, while the temperate region in the south presented the lowest prevalence values. This study offers the first report of dogs seropositive for the Costa Rican genotype in Brazil.

The detection of Fasciola eggs in ruminant fecal samples typically occurs around 15 weeks post-infection during patent infections. The recent introduction of coproantigen ELISA (cELISA) diagnostics, which capture antigenic enzymes in fecal material, holds promise for monitoring Fasciola fecal egg count (FFEC) to implement control programs, particularly targeted treatments. This study compared two study groups, those with positive FFEC (FFEC+ve) and negative FFEC (FFEC-ve), to assess the sensitivity of Flukefinder sedimentation and a cELISA kit. This study reports that the coproantigen ELISA detection limit was 4.5 eggs per gram (epg) for a 100% positivity rate. Additionally, a moderate, statistically significant positive correlation (r²=0.716, p-value < 0.01) and an odds ratio of 1.96 (p-value < 0.01) were observed between FFEC and Fasciola coproantigen from cELISA. This implies that for every single unit increase in FFEC, there is a 96% likelihood of obtaining a higher optical density (OD) reading from the cELISA test. The results suggest that cELISA can complement fecal sedimentation tests, serving as a valuable tool for detecting cattle with high FFEC. This capability facilitates targeted treatment strategies, thereby aiding in the control and prevention of the spread of fasciolosis within the studied area.

The aim of this study was to analyze the profile of IgG subclasses in COVID-19 convalescent Puerto Rican subjects and compare this profile with those exhibited by no-infected immunocompetent or immunocompromised subjects that received the mRNA vaccine. The most notable find-ngs from this study are: 1) Convalescent subjects that were not hospitalized developed high and long-lasting antibody response. 2) Both, IgG1 and IgG3 are the subclasses more prevalent in the SARS-CoV-2 infected population whereas IgG1 is more prevalent after vaccination. 3) Individuals that first had the infection and further received two doses of mRNA vaccines exhibited more robust neutralizing capacity against Omicron than those that were never infected and received two doses of Pfizer-BioNTech vaccine. 4) A class switch toward the “anti-inflammatory” anti-body isotype IgG4 is induced a few weeks after the third dose, which peaked abruptly and remained at high levels for a long period. Moreover, the high levels of IgG4 are concurrent with high neutralizing percentages against various VOC including Omicron. 5) Subjects with IBD also develop IgG4 after the third dose, although these antibody levels have limited effect on the neutralizing capacity. Knowing that the mRNA vaccines do not prevent reinfections, all Omicron sub-variants that are dominant worldwide have shown to be less pathogenic, and the IgG4 levels have been associated to immunotolerance and causative of numerous negative effects, the recommendations of successive administration of booster vaccinations to people should be revised.

The use of antibiotics grown promoter has been banned, due led problems with drug residues in animal products and increased bacterial resistance. For several reasons there is a growing interest in the scientific community in immunoglobulin Y as antibiotic alternative and their oral administration in the polyclonal antibody (pAb) format, to maintain animal health and performance, do not require IgY purification for large-scale production, resulting in protein impurities and high concentration of biotin in the samples. The signal amplification through non-covalent interaction of biotin with streptavidin has been extensionally used by many laboratories for several diagnosis diseases and scientific research in enzyme-linked immunosorbent assay (ELISA). However, the riches sources of biotin as egg yolk, leading to high biotin concentrations in samples, harming the accuracy of diagnostic and proteins concentrations tests. This study aimed to evaluate the biotin influence on measurement of Immunoglobulin Y in egg yolk freeze dried samples from immunized laying hens by immunoassays using biotin-avidin/streptavidin. The IgY concentration changed down in immunoassays using biotin-avidin/streptavidin. The detection of IgY in yolk samples by ELISA using streptavidin–biotin binding as part of the assay methodology requires some technology to neutralize high concentration of biotin on sample or more steps beyond delipidation to isolate the target protein. Otherwise, an ELISA without the use of streptavidin-biotin binding would be more advisable to avoid biotin and targets protein relationship and prevent biotin interference on results.

There is still an ambiguous opinion on oat regarding its safety for people with celiac disease (CD). Some studies have confirmed different content of oat immunoreactive epitopes in different cultivars while others explain the differences in consequence of cross-contamination with gluten-rich species or as ELISA cross-reactivity of oat homological epitopes with antibodies against wheat gliadin. Our study was based on a two-year mapping of oat immunoreactive epitopes in a set of 132 oat cultivars using the G12-based ELISA kit. Although repeated evaluation confirmed high interannual variability (RSD ≥ 30%) in approximately 2/3 of the cultivars, the permitted gluten content (20 mg kg-1) has not been exceeded except for contaminated cultivar Sirene. The polymorphism of purified avenins determined by SDS-PAGE revealed the occurrence of 2 bands around 30 kDa in oat cultivars with relatively high gluten content (12-16 mg kg-1) except for the cultivar Leo while this pattern occurred only in 50% oat cultivars with low gluten content. Quantification of gluten epitopes on purified avenin unit further revealed the three materials (Mojacar, Maris Oberon, SG-K 16370) with the lowest gluten content and presence of one band at 30 kDa. The band pattern at 30 kDa thus represents a promising breeding marker.

Ten-eleven translocation (TET) enzymes are implicated in DNA demethylation through dioxygenase activity, which converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC). However, the specific roles of TET enzymes and 5-hmC levels in head and neck squamous cell carcinoma (HNSCC) have not yet been evaluated. In this study, we analyzed 5-hmC levels and TET mRNA expression in a well-characterized dataset of 117 matched pairs of HNSCC tissues and normal tissues. 5-hmC levels and TET mRNA expression were examined via enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. 5-hmC levels were evaluated according to various clinical characteristics and prognostic implications. Notably, we found that 5-hmC levels were significantly correlated with tumor stage (P = 0.032) and recurrence (P = 0.018). Univariate analysis revealed that low levels of 5-hmC were correlated with poor disease-free survival (DFS; log-rank test, P = 0.038). The expression of TET family genes was not associated with outcomes. In multivariate analysis, low levels of 5-hmC were evaluated as a significant independent prognostic factor of DFS (hazard ratio: 2.352, 95% confidence interval: 1.136–4.896; P = 0.021). Taken together, our findings showed that reduction of TET family gene expression and subsequent low levels of 5-hmC may affect the development of HNSCC.

Effects of pulsed electromagnetic fields (PEMF) on immunological factors in a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD) model were investigated. Hairless mice were randomly assigned to control, acetone and olive oil solution-treated (AOO), PEMF 15 Hz, PEMF 75 Hz, and sham groups (n=5 each). AOO solution was used to dissolve DNCB. Both PEMF and sham groups were exposed to similar DNCB doses, causing similar AD symptoms. After AD induction for five weeks, only the PEMF group was exposed to PEMF stimulations (15 Hz, 75 Hz, and 15 mT) for two weeks inside the solenoid coil. In both groups, splenomegaly was observed, as AD was induced by hyperimmune reactions caused by DNCB sensitization. However, splenomegaly did not occur in the PEMF-exposed groups, and spleen weight decreased similarly to that of the control. Hence, the total splenocytes in the PEMF group were similar to those in the control group, whereas the sham group showed thrice the number of splenocytes compared to the PEMF group. The serum immunoglobulin E levels did not significantly change in the PEMF group; however, they increased more than fourfold in the sham group. These results demonstrate that PEMF stimulation ameliorates the abnormal symptoms caused by hyperimmune reactions.

Today, given the paramount importance of food safety to human health, mycotoxins are considered especially important contaminants of foodstuffs. Among the mycotoxins, aflatoxins are potent food contaminants which are present in maize and wheat. Invasions of toxigenic fungi in the field and during storage are directly affected by climate and other environmental factors. A total of 129 samples of maize and grain harvested in 2022 were surveyed for mycological contamination. Aflatoxin B1 contamination was investigated using the enzyme-linked immunosorbent assay (ELISA) method.Our study revealed the presence of fungi from five genera: Aspergillus, Fusarium, Penicillium, Alternaria, and Cladosporium. We also found that maize was more frequently contaminated than wheat. Fungi of the Penicillium genus were the most abundant (77.89%), followed by Fusarium (74.73%) and Aspergillus (72.63%). Samples originating from the Korça region (10⁴ units) exhibited a higher fungal load on the three genera. AFB1 contamination is a more critical issue in maize than wheat. The incidence of AFB1 in maize was 88.23%, with a maximum concentration of 69.12 μg/kg; in contrast, the incidence of AFB1 in wheat was only 4.91%. In wheat, no samples had AFB1 concentrations above the EU Maximum Residue Level (MRL) of 2 μg/kg; in maize, 41.18% of samples exceeded the AFB1 MRL of 5 μg/kg for human food, and 32.25% exceeded the MRL for animal feed. These findings, together with other research results from the last decade, suggest that relevant information should be made available to farmers, and good agriculture practices encouraged, as a matter of priority.

Aujeszky Disease Virus (ADV) is a double-stranded DNA virus with a lipoprotein envelope. The natural hosts of the infection are suidae, but the virus can infect many other mammals. The gold-standard method identified by the WOAH for the diagnosis of Aujeszky disease is ELISA method. The objective of this study was to compare the performance of meat juice and oral fluid matrices using a commercial ELISA kit designed for serum. A total of 80 blood and oral fluid samples were collected from four pig farms selected for this study. Diaphragm muscle samples of about 100 g and blood samples were collected from 213 animals at the abattoir. These biological matrices were collected from the same animals and tested using a competitive ELISA kit to detect antibodies against ADV. The relative accuracy of the meat juice compared to that of the serum was 96.7% (95% CI: 93.3%-98.7%), with 206 correct results out of 213. The relative accuracy of the oral fluid compared to that of the serum was 61.3% (95% CI: 49.7%-71.9%), with 58 correct results out of 80. Meat juice has a better combination of sensitivity and specificity than oral fluid. The usage of meat juice in routine diagnostic examinations could be achieved after further investigations to optimize the procedure.

This study is aimed to point out the possible application of the Enzyme-Linked ImmunoSorbent Assay (ELISA) test on table olives. Because olives, like many other foods, are not free from mycotoxin contamination, the main focus of this work was assessing the presence or absence of aflatoxins and ochratoxin A. The work was carried out in two phases. In the first phase, tests were carried out on commercial olives to assess the efficacy of the method and the kit (Veratox® 'Neogen' and AgraQuant® 'Romer Labs') by comparing them with the Liquid Chromatography coupled to a mass-mass spectrometer (LC-MS/MS); in order to understand if there were any interferences in antigen-antibody recognition; subsequently, mycotoxins were inoculated on the same samples with a known quantity, in order to assess the concentrations obtained and compare them to the expected ones. The second step involved modifying the method, so to improve the recovery of inoculated mycotoxins in the samples under study. Afterwards we evaluated the presence of mycotoxins in table olives supplied in the project. The results showed the effectiveness of the ELISA test, especially in the mycotoxin recovery phase. Indeed, after the modification of the mycotoxin extraction method, recoveries increased from 55% to over 70% for aflatoxins, and from 40% to over 55% for ochratoxin A. When analysing the samples provided by the above-mentioned company, the results showed some false positives, in particular in relation to ochratoxin A ’s analysis, mainly related to the first sample submission. Taking this into account, the ELISA test can be considered as an initial screening method applicable as a control programme for the evaluation of mycotoxins in olives which helps by saving time and costs. It should always be kept in mind that, if positive values should emerge, the LC-MS/MS analysis should be carried out.

Background: MERS-CoV is a zoonotic virus that have emerged in humans in 2012 and caused severe respiratory illness with mortality rate of 34.4%. Since its appearance, MERS-CoV have been reported in 27 countries and most of these cases were in Saudi Arabia. So far, dromedaries are considered to be the intermediate host and the only known source of human infection. Method: This study was designed to determine the seroprevalence and the infection rate of MERS-CoV in slaughtered food-camels in Riyadh, Saudi Arabia. A total of 171 nasal swabs along with 161 serum samples were collected during the winter; from January to April 2019. Nasal swabs were examined by Rapid test and RT-qPCR to detect MERS-CoV RNA, while serum samples were tested primarily using S1-based ELISA Kit to detect MERS-CoV (IgG) antibodies and subsequently by MERS pseudotyped viral particles (MERSpp) neutralization assay for confirmation. Genetic diversity of the positive isolates was determined based on the amplification and sequencing of the spike gene. Results: Our results showed high prevalence (38%) of MERS-CoV infection in slaughtered camels and high seropositivity (70.81%) during the time of the study. These data indicate previous and ongoing MERS-CoV infection in camels. Phylogenic analysis revealed relatively low genetic variability among our isolated samples. When these isolates were aligned against published spike sequences of MERS-CoV, deposited in global databases, there was sequence similarity of 94%. Conclusion: High seroprevalence and high genetic stability of MERS-CoV in camels indicating that camels pose a public health threat. The widespread of MERS-CoV infections in camels increases the risk of future zoonotic transmission into people with direct contact with these infected camels. This study confirms re-infections in camels, highlighting a challenge for vaccine development when it comes to protective immunity.

This study was conducted to investigate the prevalence of Coxiella burnetii infection according to cattle breeds and growth types. A total of 491 cattle [cattle breed: 216 dairy cattle and 275 beef cattle; according to growth type: indoor housing (n = 294) and grazing (n = 197)] were tested for the presence of C. burnetii DNA and antibodies against C. burnetii using a commercial enzyme-linked immunosorbent assay (ELISA). Overall, 10.8% and 8.8% of the cattle were positive by PCR and for C. burnetii antibodies, respectively. The prevalence of C. burnetii was significantly higher in beef cattle than in dairy cattle using PCR (13.6% vs 7.4%; P = 0.032) and ELISA (14.6% vs 1.4%; P = 0.000), respectively. The overall infection rate of C. burnetii was significantly high in grazing cattle (PCR: 24.9%, ELSIA: 21.3%; P = 0.000) compared with housing cattle (PCR: 1.4%, ELISA: 0.3%). The results indicate that beef cattle have a significantly higher risk of contracting C. burnetii infection compared with dairy cattle (21.5% vs. 7.9%, χ2 = 5.82, P = 0.000, odds ratio = 3.197, 95% CI: 1.80-5.67). In addition, the infection of C. burnetii was significantly associated with grazing (P = 0.000). Moreover, a risk of contracting C. burnetii infection in grazing cattle was increased by 32.57-fold (95% CI: 12.84-82.60, P = 0.000) compared with indoor housed cattle. The phylogenetic analysis based on the IS111 gene revealed that our isolates were grouped together with humans, ticks, goats, and cattle isolates found in several countries. C. burnetii isolates circulating in the Republic of Korea exhibit genetic variations. Consequently, our results suggest that cattle are potential reservoirs for C. burnetii infection and most importantly, grazing acts as a high risk factor for the occurrence and transmission of this infection.

Lumpy skin disease (LSD) is a viral disease of cattle and water buffalo characterized by cutaneous nodules, biphasic fever and lymphadenitis. LSD is endemic in Africa and Middle East but in recent years has spread in different Asian countries. Drop of animal productions, vaccination campaign, surveillance costs and trade restrictions have significant impact on the economy of affected countries. The disease is well characterized in cattle while little is known in buffaloes and no experimental studies have been conducted in this species. Six buffaloes and two cattle were inoculated with an Albanian LSD virus (LSDV) field strain. Over a period of 42 days animals were clinically monitored and sampled for serological and virological analysis. Only two buffaloes showed clinical sign, fever, skin nodules and lymphadenitis. All samples tested in real time PCR (blood, swabs, biopsies and organ) gave negative results. Three buffaloes seroconverted at day 39 and 42 post-inoculation, detected by ELISA, but all sera were negative in serum neutralization test. Cattle showed more severe clinical signs. Moreover, viraemia and virus shedding were proven by positive PCR results and antibodies were detected by both ELISA and serum neutralization test. These findings demonstrated the limited susceptibility of buffaloes compared to cattle once experimental infected with LSDV. Further studies are needed to investigate the epidemiological role of the buffaloes.

Background: Tetanus is an acute, fatal disease caused by exotoxins released from Clostridium tetani during infections. A protective humoral immune response can be induced by vaccinations with pediatric and booster combinatorial vaccines that contain inactivated tetanus neurotoxin (TeNT) as a major antigen. Although some epitopes in NeNT have been described using various ap-proaches, a comprehensive list of its antigenic determinants that are involved with immunity have not been elucidated. To that end, a high resolution analysis of the linear B-cell epitopes in NeNT was performed using antibodies generated in vaccinated children. Methods: Two hundred sixty-four peptides that cover the entire coding sequence of TeNT protein were prepared in situ on a cellulose membrane by SPOT synthesis and probed with sera from children vaccinated (ChVS) with a triple DTP-vaccine to map continuous B-cell epitopes that were further characterized and validated by immunoassays. Results: Forty-three IgG epitopes were identified. Four (TT-215-218) were chemically synthesized as Multiple Antigen Peptides (MAPs) and used in peptide ELISAs to screen post-pandemic DTP vaccinations. The assay displayed a high performance with high sensitivity (99.99%) and specificity (100%). Conclusions: The complete map of linear IgG epitopes induced by vaccination with inactivated TeNT highlights three key epitopes involved in the ef-ficacy of the vaccine. Antibodies against epitope TT-8/G can block enzymatic activity, and those against epitopes TT-40/G and TT-42/G can interfere with TeNT binding to neuronal cell receptors. We further show that four of the epitopes identified can be employed in peptide ELISAs to assess vaccine coverage. Overall, the data suggest a set of select epitopes to engineer new, directed vaccines

The apicomplexan parasite Theileria haneyi is one of two known causative agents of equine theileriosis. It causes milder clinical disease than its more virulent counterpart, Theileria equi, in experimentally infected horses, and can superinfect T. equi-positive horses. The current EMA1-based competitive ELISA used in the U.S. to detect equine theileriosis detects T. equi but not T. haneyi, and the complexity of molecular assays precludes widespread use for epidemiologic studies. In order to facilitate urgently needed studies on the prevalence of T. haneyi, the goal of this study was to develop a sensitive and specific serologic assay for the diagnosis of T. haneyi based on the equi mero-zoite antigen 11 (ThEMA11). To achieve this objective, ThEMA11 was recombinantly expressed in eukaryotic cells and its antigenicity assessed using sera from T. haneyi-experimentally infected horses. Confirmation of sera reactiv-ity enabled design and optimization of an indirect ELISA. Specificity of the ELISA for T. haneyi was assessed using a cohort of sera from horses experimentally infected and confirmed PCR-positive for either T. equi or T. haneyi. Data from field samples further demonstrate that the ThEMA11 ELISA is capable of identifying T. haneyi antibodies in horses from multiple continents around the world.

As the coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses. The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

Adlay seed samples were collected from 3 adlay growing regions (Yeoncheon, Jeonnam and Eumseong regions) in Korea during 2012. Among all the samples collected, 400 seeds were tested for fungal occurrence by standard blotter and test tube agar methods and different taxonomic groups of fungal genera were detected. The most predominant fungal genera encountered were Fusarium, Phoma, Alternaria, Cladosporium, Curvularia, Cochliobolus and Leptosphaerulina. The occurrence of Fusarium species were 45.6% and based on the combined sequences of two protein coding genes, EF-1a, Beta-tubulin and phylogenetic analysis, 10 species were characterized as F. incarnatum (11.67%), F. kyushense (10.33%), F. fujikuroi (8.67%), F. concentricum (6.00%), F. asiaticum (5.67%), F. graminearum (1.67%), F. miscanthi (0.67%), F. polyphialidiom (0.33%), F. armeniacum (0.33%) and F. thapsinum (0.33%). The ability of these isolates to produce mycotoxins fumonisin (FUM) and zeralenone (ZEN) were tested by ELISA quantitative analysis method. The result revealed that fumonisin (FUM) was produced only by F. fujikuroi and zeralenone (ZEN) by F. asiaticum & F. graminearum. Mycotoxigenic species were then examined for their morphological characteristics to confirm their identity. Morphological observations of the species correlated well with their molecular identification and confirmed as F. asiaticum, F. fujikuroi and F. graminearum.

Hepatitis E virus (HEV) is a zoonotic foodborne virus with an annual infection prevalence of 20 million human cases, which seriously affects public health and economic development in both developed and developing countries. To better understand the epidemiology of HEV in the Centre of Portugal, a cross-sectional study was conducted from 2016 to 2023 with sera samples from wild ungulates. The seroprevalence and risk factors for HEV seropositivity were evaluated in the present study. Specifically, antibodies against HEV were determined by a commercial enzyme-linked immune-sorbent assay (ELISA). Our results show that in the 650 sera collected from 298 wild red deer and 352 wild boars from Portugal, 9.1% red deer (95% confidence interval [CI]: 6.312.9%) and 1.7% wild boar (95% CI: 0.63.3%) were positive for antibodies to HEV. Regarding age, the seropositivity in juvenile wild ungulates was 1.3% (95% CI: 0.273.72%) and 7.2% in adults (95% CI: 4.910.11%). Logistic regression models investigated risk factors for seropositivity. The odds of being seropositive was 3.6 higher in adults than in juveniles (95% CI: 1.7218.11%) and the risk was 4.2 higher in red deer than in wild boar (95% CI: 1.64–10.69%). Both wild ungulate species were exposed to HEV. The higher seroprevalence in red deer suggests that this species may have a major contribution to the ecology of HEV in the Centre of Portugal. Further research is important to understand how wildlife affects the epidemiology of HEV infection in Portugal.

Wheat is one of the global strategic crops and ranks third in terms of cereals production. Wheat crop is exposed to many fungal infections during the cultivation stages, some of which have the ability to secrete numbers of toxic secondary metabolites that threaten the quality of grains, consumer health, producer economics and global trade exchange. Fifty-four random samples were collected from wheat which originated from different countries. The samples included 14 of soft wheat to study the extent of their contamination with Deoxynivalenol (DON) and T-2 toxin by auto-ELISA technology and r-biopharm Microtiter plate. All samples were contaminated with DON toxin except one sample, and the contamination average ranged between 40.7 and 1018.8 µg kg-1. The highest contamination rates were in Lithuanian and the lowest in Indian wheat. Meanwhile the highest average level of T-2 toxin contamination was in Lithuanian wheat grains with a rate of 377.4µg kg-1 and the lowest in Polish wheat with an average of 115.3µg kg-1. GC-MS/MS and multiple reaction monitoring mode (MRM), was used to detect 15 triazole derivatives in collected samples, which may be used to combat fungal diseases on wheat during the growing season. Only 9 derivatives were found; Simeconazole, Penconazole, Hexaconazole, Cyproconazole, Diniconazole, Tebuconazole, Metconazole, Fenbuconazole and Difenoconazole. These derivatives varied according to the origin of wheat samples as well as their its concentration whereas, another 6 derivatives were not detected in all samples. A direct inverse relationship was found between the DON concentration in the samples and the residues of Simeconazole, Penconazole, Diniconazole, Tebuconazole, Metconazole, Fenbuconazole and Difenoconazole and the T-2 toxin showed the same relationship except for Tebuconazole. The safe and rational use of some triazole derivatives may be a new approach and a promising strategy not only to reduce plant diseases and their problems, but also to get rid of some mycotoxins as contaminates.

The ongoing pandemic of COVID-19 has not only commenced a global health emergency but agitated various aspects of humanity. During this period of crisis researchers over the world have ramped their efforts to constrain the disease in all possible ways whether it is vaccination, therapy, or diagnosis. Since the spread of the disease has not yet elapsed sharing the ongoing research findings could be the key to disease control and management. An early and efficient diagnosis could leverage the outcome until a successful vaccine is developed. Molecular tests both in-house and commercial kits are preferably being used worldwide in the COVID-19 diagnosis. However, the limitation of high prices and lengthy procedures impede their use for mass testing. Keeping the constant rise of infection in mind search for an alternative test that should be cost-effective, simple, and suitable for large scale testing and surveillance is a need of an hour. One such alternative could be the immunological tests. Therefore, in the last few months deluge of immunological rapid tests has been developed and validated across the globe. The objective of the present review is to share the diagnostic performance of various immunological assays reported so far in SARS-CoV-2 case detection. The article consolidated the studies (published and preprints) related to the serological tests such as chemiluminescence, enzyme-linked and lateral flow-based point-of-care tests in COVID-19 diagnosis and updated the current scenario. This review will hopefully be an add-on in COVID-19 research and will contribute to congregate the evidence for decision-making.

For the diagnosis of disease, the ability to quantitatively detect trace amounts of the causal proteins from bacteria/viruses as biomarkers in patient specimens is highly desirable. Here we introduce a simple, rapid, and colorimetric assay as a de novo, ultrasensitive detection method. This ultrasensitive assay consists of sandwich enzyme-linked immunosorbent assay (ELISA) and thionicotinamide-adenine dinucleotide (thio-NAD) cycling, forming an ultrasensitive ELISA, in which the signal substrate (i.e., thio-NADH) accumulates in a triangular manner, and the accumulated thio-NADH is measured at its maximum absorption wavelength of 400 nm. We have successfully achieved a limit of detection of ca. 10–18 moles/assay for a target protein. As an example of infectious disease detection, HIV-1 p24 could be measured at 0.0065 IU/assay (i.e., 10⁻¹⁸ moles/assay), and as a marker for a lifestyle-related disease, adiponectin could be detected at 2.3 × 10⁻¹⁹ moles/assay. In particular, despite the long-held belief that the trace amounts of adiponectin in urine can only be detected using a radioisotope, our ultrasensitive ELISA was able to detect urinary adiponectin. This method is highly versatile, because simply changing the antibody enables the detection of various proteins. This assay system requires only the measurement of absorbance, thus it requires equipment that is easily obtained by medical facilities, which facilitates diagnosis in hospitals and clinics. Moreover, we describe an expansion of our ultrasensitive ELISA to a non-amplification nucleic acid detection method for nucleic acids using hybridization. These de novo methods will enable simple, rapid, and accurate diagnosis.

Keywords: COVID-19, SARS-CoV-2, Antibody, ELISA, Spike, S1, S2, RBD, epitopes, Neutralization

Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 Phage Display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations.

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the his-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.

The study aimed to manage and to analyse the results of the laboratory tests, routinallypractice for hepatitis C diagnosis, using blood tests. Statistical analysis of this study results, was performed using the laboratory informatic system. The results of the study are substantial and intricate reffering to comparision between two methods concretlly ELISA method (Enzyme-Linked Immunosorbent Assay) and chemiluminescence methods and their results. Good to know our opinion that RT-PCR technique, it is considered proper for the diagnosis of HCV ( Hepatitis C virus).

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modified putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 to 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Scrub typhus is a life-threatening infectious disease and always creating a diagnostic dilemma in terms of rapid turnaround time and accuracy, qRT PCR can become a very good option to achieve the desired result with the molecular level of accuracy and boost up the rapid patient management. This study was performed to evaluate the performance of qRT PCR in comparison to commonly used IgM ELISA and Weil-Felix tests to diagnose scrub typhus, as well as to look for the demographic and clinical profile of the disease in North-East India. It was a hospital-based prospective study conducted in a tertiary care hospital of north-east India, over a period of 1 year, in which all the samples from suspected scrub typhus cases were screened by Weil-Felix test as per institute’s diagnostic protocol after which IgM ELISA for Scrub Typhus was performed. All the IgM positive samples and 20 highly suspected but ELISA negative samples were subjected to qRT PCR, targeting 56 kDa type-specific gene of O. tsutsugamushi. Statistical analysis was done by MS-Excel for Windows v2013® and MedCalc® v17.9 for Windows (MedCalc Software, Acacialaan 22, B-8400 Ostend, Belgium). In this study, we have successfully evaluated the performance of qRT PCR kit for diagnosis of scrub typhus. Out of 54 samples tested, 24 IgM ELISA positive samples and 3 IgM ELISA negative samples have shown the presence of bacterial DNA with quantification of DNA copies. It has also been observed that 21 out of 27 PCR positive samples (77.8%) were detected within the 1st 7 days of illness. All the demographic, as well as clinical data, were also analysed. The performance of the commercial qRT PCR kit used in our study is satisfactory, which provides the extra advantage of quantification of DNA copies and increases diagnostic accuracy within the 1st week of fever.

Human-to-animal SARS-CoV-2 transmission was observed, including a veterinarian contracting COVID-19 through close contact with an infected cat, suggesting an atypical anthropozoonotic transmission. This study investigated the prevalence of SARS-CoV-2 antibodies in cats during human outbreaks and elucidated the correlation between cat infections and human epidemics. A total of 1,107 cat sera samples were collected and screened for SARS-CoV-2 antibodies using a modified indirect ELISA human SARS-CoV-2 antibodies detection kit. The samples were confirmed using a cPass surrogate viral neutralization assay. The SARS-CoV-2 seropositivity rate was 22.67% (199/878), mirroring the trend observed in concomitant human case numbers. The waves of an epidemic and provinces did not significantly impact ELISA-positive cat. Notably, Chon Buri ex-hibited a strong positive correlation (r = 0.99, P = 0.009) between positive cat sera and reported human case numbers. Additionally, cPass surrogate viral neutralization revealed a 3.99% (35/878) seropositivity rate. While there were significant differences in positive cat numbers and proportions between epidemic waves. In Samut Sakhon, a positive correlation (r = 1, P = 0.042) was noted between the proportion of positive cat sera and reported human cases. The findings emphasize the need for ongoing surveillance to comprehend SARS-CoV-2 dynamics in both human and feline populations.

Global severity maps of ongoing dengue epidemic and COVID-19 pandemic do not overlap. Countries with high dengue endemicity (>1.5 million cases/year) are observably less hit by COVID-19 in terms of infection, transmission and mortality. Based on non-overlap of dengue and COVID-19 severity maps and evidence of SARS-CoV-2 serological cross-reactions with dengue, we wonder whether immediate immunization of susceptible populations in Europe, North America and Asia (China, Iran) with available live-attenuated dengue vaccines, will cue the anti-viral immune response to thwart COVID-19 (viral interference). Risk of developing post-vaccination “Antibody-dependent Enhancement” is low as dengue is not endemic in the aforesaid regions.

The Developing Countries Vaccine Manufacturing Network coordinated a multi-laboratory study funded by the US National Institute for Innovation in Manufacturing Biopharmaceuticals to assess the performance of the Pertussis Serological Potency Test (PSPT) in the laboratories of seven whole-cell pertussis vaccine (wP) manufacturers and three National Control Laboratories based in Southeast Asian countries. To this end, common protocols for the immunization of mice and serological testing by ELISA were shared with all laboratories. Each laboratory used their own wP vaccines and the reference vaccine routinely used in the regulatory required mouse protection test (MPT) and assayed the products both in the MPT and PSPT. Whereas in all laboratories the PSPT showed decreased serum IgG levels after immunization with sub-potent compared to potent final batches, some issues were encountered during execution of the assay, which prevented drawing of definitive conclusions on assay performance. Nevertheless, the study provided the participants with the opportunity to familiarize themselves with the PSPT protocols and revealed the next steps to be addressed for implementation of the method.

The enzyme-linked immunosorbent assay (ELISA) coupled with surface-enhanced Raman spectroscopy (SERS) detection is a growing area of analytical chemistry emerged as a solution to a problem of sensitivity improvement. SERS-based ELISA with horseradish peroxidase as an enzymatic label, o-phenylenediamine (oPD) substrate, and 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such systems. Surprisingly, the full potential of this long-known approach has not been revealed yet. The current study addresses a previously neglected problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of pH, concentration of aggregating agent, and particle surface chloride stabilizer were extensively evaluated. At optimal mildly acidic pH of 3, 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, at least two orders of magnitude advantages in the detection limits of SERS over colorimetry both for DAP and HRP were demonstrated. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Autoantibodies against type 1 interferons (IFN-I) are highly specific marker for type 1 autoimmune polyglandular syndrome (APS-1). Moreover, determination of antibodies to IFN-ω and IFN-α2 allows a short-term diagnosis in patients with isolated and atypical forms of APS-1. In this study, a comparison of three different methods, namely, multiplex microarray-based, cell-based and enzyme-linked immunosorbent assays for detection of antibodies against omega-interferon and alpha2-interferon was carried out. A total of 206 serum samples from adult patients with APS-1, APS-2, isolated autoimmune endocrine pathologies, non-autoimmune endocrine disorders and healthy individuals were analyzed. In the APS-1 patient cohort (n=18), there was good agreement between the results of anti- IFN-I antibody tests performed by three methods, with 100% specificity and sensitivity for microarray-based assay. Although only the cell-based assay can determine the neutralizing activity of autoantibodies, the microarray-based assay can serve as a highly specific and sensitive screening test to identify anti- IFN-I antibody positive patients.

Accurate diagnosis at an early stage of infection is essential for the successful management of any contagious disease. The COVID-19, caused by the SARS-CoV-2 virus is a pandemic that has affected 214 countries affecting more than 30.8 million people causing 0.957 million deaths as of third week of September, 2020. The primary diagnosis of the infection is done either by the molecular technique of RT-qPCR by detecting portions of the RNA of the viral genome or through immunodiagnostic tests by detecting the viral proteins or the antibodies produced by the host. As the demand for the test increased rapidly many naive manufacturers entered the market with novel kits and more and more laboratories also entered the diagnostic arena making the test result more error-prone. There are serious debates globally and regionally on the sensitivity and specificity of these tests and about the overall accuracy and reliability of the tests for decision making on control strategies. The significance of the test is also complexed by the presence of asymptomatic carriers, re-occurrence of infection in cured patients as well as by the varied incubation periods of the infection and shifting of the viral location in the host tissues. In this paper, we review the techniques available for SARS-CoV-2 diagnosis and probable factors that can reduce the sensitivity and specificity of the different test methods currently in vogue. We also provide a check-list of factors to be taken care to avoid fallacious practices to reduce false positive and false negative results by the clinical laboratories

Since its 2013 emergence in the Americas, chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a Ct cutoff of 22. As the Ct value increased from 22, ELISA sensitivity decreased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe is becoming increasingly important due to their close proximity to humans, the increasing import of dogs from endemic regions and impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA was compared with three rKLi8.3 based diagnostic test systems, the rKLi8.3 ELISA, the INgezim® Leishma CROM lateral flow test (LFT) and the VetBlot® Leishmania LineBlot. CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo-and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens, showed a very high specificity for healthy dogs from endemic regions of both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, sero-diagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens and the LFT has the advantage of providing a laboratory independent, rapid and specific diagnosis of CanL.

Contradictory reports are available on vaccine-associated hyperstimulation of the immune system, provoking the formation of pathological autoantibodies. Despite being interconnected within the same network, the role of the quieter, yet important non-pathological; natural autoantibodies (nAAbs) is less defined. We hypothesize that upon a prompt immunological trigger, also physiological nAAbs exhibit a moderate plasticity. We investigated their inducibility through aged and recent antigenic triggers. Anti-viral antibodies (anti-MMR; n=1739 and anti-SARS-CoV-2 IgG; n=330) and nAAbs (anti-citrate synthase IgG, IgM; n=1739) were measured by in-house and commercial ELISAs, using Croatian (Osijek) anonymous samples with documented vaccination background. Results were subsequently compared for statistical evaluation. Interestingly, IgM isotype nAAb showed a statistically significant connection with anti-MMR IgG seropositivity (p< 0.001 in all cases), while IgG isotype nAAb levels were elevated in association with anti-SARS CoV-2 specific seropositivity (p= 0.019) and in heterogeneous vaccine regimen recipients (unvaccinated controls; vector/mRNA vaccines p= 0.002). Increasing evidence supports the interplay between immune activation and the dynamic expansion of nAAbs. Consequently, further questions may emerge regarding the ability of nAAbs silently shaping the effectiveness of immunization. We suggest re-evaluating the impact of nAAbs on the complex functioning of the immunological network.

In contrast to normal cells, tumor cells of multiple entities overexpress the Heat Shock Protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane-Hsp70 positive tumor cells actively release Hsp70 into lipid microvesicles termed exosomes into the blood. Due to conformational changes of Hsp70 in the lipid environment, most commercially available antibodies fail to detect membrane-bound and exosomal Hsp70. To fill this gap and to assess the role of exosomal Hsp70 in the circulation as a potential tumor biomarker, we established the novel complete Hsp70 (compHsp70) sandwich ELISA using two monoclonal antibodies (mAbs) that are able to recognize both, free and lipid-associated Hsp70 on the cell surface of viable tumor cells and exosomes. The epitopes of the mAbs cmHsp70.1 (aa 451-461) and cmHsp70.2 (aa 614-623) that are conserved among different species reside in the substrate-binding domain of Hsp70, with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/ml, high recovery rates of ‘spiked’ liposomal Hsp70 (>84%), comparable values between human serum and plasma samples, and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy volunteers. Hsp70 concentrations dropped concomitantly with the decrease in viable tumor mass on irradiation of patients with approximately 20 Gy (range 18 – 22.5 Gy) or after completion of radiotherapy (60 - 70 Gy). In summary, the compHsp70 ELISA presented herein provides a highly sensitive and reliable tool for measuring free and exosomal Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a predictive tumor-specific biomarker, risk assessment and for monitoring therapeutic outcome.

Proteomics in respiratory allergic diseases has such a battery of techniques and programs that one would almost think that there is nothing impossible to find, invent or mold. All these resources that we document here, are involved in solving problems in allergic diseases, both diagnostic, prognostic, treatment and immunotherapy development. The main perspectives, according to this version, are in three strands and / or lockout immunological system: 1) In blocking the diapedesis of cells involved, 2) modifications and blocking of paratopes and epitopes being understood by modifications to antibodies, antagonism or blocking them and 3) blocking FcεR1 receptors to prevent specific IgEs from sticking to mast cells and basophils. These tools and targets in the allergic landscape are, in our view, the prospects in the field.

Keywords: Pulp cell, Periodontal cell, Micro-Arc Oxidation (MAO), Sever Plastic deformation (SPD), Equal Channel Angular Pressing (ECAP), AlamarBlue, ELISA, trypsin

Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by western blot, ELISA and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy-, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and 44 samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5 % and a specificity of 100 % for the certified IVD. There were 7 samples with an uncertain outcome. Our VLP-ELISA showed superior performance with a sensitivity of 97.5 %, a specificity of 100 %, and only 3 uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

Prostate cancer (PC) is the second most diagnosed cancer among men. It was observed that early diagnosis of disease is highly beneficial for survival of cancer patients. Therefore, extension and increasing quality of life of PC patients can be achieved by broadening the Cancer Screening programs that are aimed at the identification of cancer manifestation in patients at earlier stages before they demonstrate well-understood signs of the disease. Therefore, there is an urgent need in standard, sensitive, robust, and commonly available screening and diagnosis tools for identification of early signs of cancer pathologies. In this respect, “Holy Grail” of cancer researchers and bioengineers for decades have been molecular sensing probes that would allow diagnosis, prognosis, and monitoring of cancer diseases by their interaction with cell-secreted and cellassociated PC biomarkers, e.g. PSA and PSMA correspondinly. At present, most PSA tests are performed at centralized laboratories using high throughput Total PSA immune analyzers which are suitable for dedicated laboratories and are not readily available for the broad health screening. Therefore, the current trend in detection of PC is developments of the portable biosensors for mobile laboratories and individual use. Phage display since its conception by George Smith in 1985 has emerged as a premier tool in molecular biology with widespread applications. This review describes how the paradigm of molecular evolution and phage display revolutionized the methods of early diagnosis and monitoring of PC.

Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies remain quite expensive in the foreseeable future. Also, nearly all commercial antibody suppliers may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De-novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF-MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies and to assign a specific reagent to a datasheet of a commercial supplier, a public database record or an antibody ID.

Rodents are known to be reservoirs of Toxoplasma gondii and keep the parasite circulation in the environment. We conducted biomonitoring to assess the role of sylvatic rodents in maintaining T. gondii and to analyse the prevalence and seroprevalence of the parasite in seven wild rodent species. Rodents were collected in our study sites (woodland and open grasslands) located in northeastern Poland and dissected. We collected brain, spleen, blood and serum samples. We applied both molecular (PCR assay, nested-PCR assay) and serological (ELISA and agglutination tests) methods to indicate the best approach for application in the biomonitoring of T. gondii in small mammals. We screened samples from 95 individuals sing PCR assays and found no T. gondii DNA. The agglutination test showed no signal. We found antibodies against T. gondii in 6 sera samples out of 74 analysed (seroprevalence = 8.11% [4.0-17.1]). Our results confirm that rodents participate in the life cycle of T. gondii as reservoirs of this parasite in the sylvatic environment. However, biomonitoring should be performed with the ELISA tests to search for T. gondii antigens, rather than a molecular approach only.

The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones.

The identification of a new virus Kiparis-144 (KiparisV) in a half-fed tick I persulcatus, removed from a person in the south of the Russian Far East, determined the goal of further research - to study its role in human pathology and the characteristics of its behavior in the tick microbiome. An ELISA study of 439 blood samples from people affected by tick bites indicated a fairly frequent (up to 16.4±1.7%) encounter of people with the KiparisV virus in the territory of natural foci of the Primorsky Territory. It has been shown that this virus, when mono-infected, can cause clinical symptoms characteristic of acute respiratory diseases. During co-infection under the influence of the KiparisV, there was an increase in the activity of Borrelia spp. with the constant production of antibodies to them only of the IgM class and clinical symptoms of Lyme disease for one year (observation period). Electron microscopy of the KiparisV found in a tick removed from a patient showed a picture of the relationship of the KiparisV (up to 100 nm in size) with unknown representatives of the tick microbiome - pathogens of a viral or bacterial nature. In a comparative aspect, a similar electron microscopic picture of the interaction of the KiparisV virus with a known pathogen of viral nature, the ectromelia virus (ECTV), is presented. The importance of the electron microscopy method in describing the tick microbiome is shown. Thus, it has been established that when co-cultivated in the system the helper-virus KiparisV probably tends to use another pathogen of the tick microbiome for its reproduction, influencing its pathogenicity.

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (K_d), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC₅₀ approaches the K_d value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to K_d. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.