Competitive ELISA Research Articles

Determination of 3-Phenoxybenzoic Acid by an Improved Enzyme-Linked Immunosorbent Assay (ELISA) by the Immobilization of Nanobodies with Streptavidin and Biotin

Nanobodies (Nbs) are single domain antibodies that are frequently utilized in enzyme-linked immunosorbent assays (ELISAs). However, a major limitation of nanobodies is their low sensitivity in capture assays. To address this issue, the combination of streptavidin and biotin can be employed to immobilize the nanobodies on plates in an oriented manner, thereby exposing their binding sites. In this study, nanobodies specific to 3-phenoxybenzoic acid (3-PBA) were biotinylated at their C-termini and immobilized onto streptavidin-coated microplates. A direct competitive ELISA was developed for the determination of 3-phenoxybenzoic acid in ovine and bovine urine. Compared to directly coating the nanobodies, the streptavidin-bridged nanobody capture-based ELISA demonstrated improved sensitivity, with a 16.6% enhancement, and a half-maximum inhibition concentration (IC50) of 2.05 ng/mL. The developed assay provided a linear range from 0.27 to 20.2 ng/mL (IC20-IC80) and a limit of detection (LOD) of 0.1 ng/mL. The average recoveries of 3-phenoxybenzoic acid spiked in the urine ranged from 82.57% to 102.75% and were consistent with values obtained by liquid chromatography – mass spectrometry (LC-MS). These findings demonstrate that the streptavidin-bridged capture nanobody-based ELISA significantly enhanced the sensitivity for the determination of 3-phenoxybenzoic acid residues in animal urine.

Elastin-like polypeptide-functionalized nanobody for column-free immunoaffinity purification of aflatoxin B1.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B1 (AFB1) was developed using an AFB1-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (Tt) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB1 as well as phase transition when the temperature rose above Tt. To establish the optimal conditions for cleaning AFB1, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods.

Evaluation of commercial quadrivalent foot-and-mouth disease vaccines against east African virus strains reveals limited immunogenicity and duration of protection

Foot-and-mouth disease virus (FMDV) causes a contagious disease (FMD) in cloven-hoofed animals. For FMD-endemic countries, vaccination is critical for controlling disease but is rarely monitored, despite substantial funds spent on vaccine purchases. We evaluated antibody responses in cattle to two commercial vaccines each containing antigens of four FMDV serotypes. Sampling was done over 360 days, with serology for each serotype performed using commercially available solid phase competition ELISAs (SPCE) and with virus neutralization tests (VNT) employing regionally relevant test viruses. A primary course of each vaccine was administered to 37 calves, some of which received a second dose after 28 days. Using new production batches of vaccines, all calves received a booster vaccination 180 days post vaccination, while 10 additional naïve calves were also vaccinated using the new batches and followed up for ∼180 days. Simple and general linear models were used to compare antibody responses which varied substantially according to vaccine, dose regime, serotype, and test, but were mostly insufficient to ensure a high likelihood of adequate or sustained probable protection. One of the vaccines administered as a two-dose primary course of vaccination was superior to other options, but even then, data trajectories from VNT responses suggested probable protection of 75 % of calves for 6 months for only one virus serotype. Calves administered with the other vaccine and those given a single primary dose developed low levels of antibodies, offering predicted likely protection lasting less than two months. Individual SPCE results were weakly correlated (r2 = 0.48) to neutralization and associated likelihoods of protection but SPCE and VNT agreed on which vaccine and dose regime performed best. Our findings highlight gaps in immunogenicity of FMD vaccines used in East Africa and reinforce the importance of independent quality control studies to evaluate and improve commercial FMD vaccines and vaccination regimes.

A Peptide Motif Covering Splice Site B in Neuroligin-1 Binds to Aβ and Acts as a Neprilysin Inhibitor.

The most common cause of dementia among elderly people is Alzheimer's disease (AD). The typical symptom of AD is the decline of cognitive abilities, which is caused by loss of synaptic function. Amyloid-β (Aβ) oligomers play a significant role in the development of this synaptic dysfunction. Neuroligin-(NL)1 is a postsynaptic cell-adhesion molecule located in excitatory synapses and involved in the maintenance and modulation of synaptic contacts. A recent study has found that Aβ interacts with the soluble N-terminal fragment of NL1. The present study aimed to elucidate the role of NL1 in Aβ-induced neuropathology. Employing surface plasmon resonance and competitive ELISA, we confirmed the high-affinity binding of NL1 to the Aβ peptide. We also identified a sequence motif representing the NL1-binding site for the Aβ peptide and showed that a synthetic peptide modeled after this motif, termed neurolide, binds to the Aβ peptide with high affinity, comparable to the NL1-Aβ interaction. To assess the effect of neurolide in vivo, wild-type and 5XFAD mice were subcutaneously treated with this peptide for 10weeks. We observed an increase in Aβ plaque formation in the cortex of neurolide-treated 5XFAD mice. Furthermore, we showed that neurolide reduces the activity of neprilysin, the predominant Aβ-degrading enzyme in the brain. Accordingly, we suggest that neurolide is the NL1-binding site for Aβ peptide, and acts as an inhibitor of neprilysin activity. Based on these data, we confirm the involvement of NL1 in the development of AD and suggest a mechanism for NL1-induced Aβ plaque formation.

Preparation of monoclonal antibody against rhoifolin and its application in enzyme-linked immunosorbent assay of rhoifolin and diosmin

Both rhoifolin and diosmin belong to flavonoids, which are widely present in citrus. Diosmin is not only used in the medical field in the world, but also used as a dietary supplement in the United States. Rhoifolin has a similar structure to diosmin and also exhibits antioxidant and anti-inflammatory properties. In this study, an anti-rhoifolin monoclonal antibody was prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established. The half-maximal inhibitory concentration (IC50) of icELISA was determined to be 4.83 ng/mL, and the detection range was 0.97–33.87 ng/mL. The results of UPLC-MS/MS and icELISA generally demonstrate consistency. Moreover, by exploiting the cross-reactivity of the antibody, diosmin in tablets can be detected by icELISA. The results demonstrate that the developed method has good accuracy, reproducibility, and broad application prospects.

Development and Validation of Serotype-Specific Blocking ELISA for the Detection of Anti-FMDV O/A/Asia1/SAT2 Antibodies.

Foot-and-mouth disease (FMD) is one of the most infectious viral transboundary diseases of livestock, which causes devastating global economic losses. Different enzyme-linked immunosorbent assays (ELISAs) are used for sero-surveillance of the foot-and-mouth disease virus (FMDV). However, more sensitive, accurate, and convenient ELISAs are still required to detect antibodies against FMDV serotypes. The primary goal of this study was to establish serotype-specific monoclonal antibody (mAb)-based blocking ELISAs (mAb-bELISAs) that would provide better performance characteristics or be equivalent in performance characteristics compared with a conventional polyclonal antibody (pAb)-based competitive ELISA (pAb-cELISA). Four mAb-bELISAs were developed using FMDV serotype-specific mAbs for the detection of anti-FMDV/O/A/Asia1/SAT2 antibodies. Using a 50% cut-off, all four mAb-bELISAs exhibited species-independent 99.74%, 98.01%, 96.59%, and 98.55% diagnostic specificity (DSp) and 98.93%, 98.25%, 100%, and 87.50% diagnostic sensitivity (DSe) for FMDV serotypes O, A, Asia1, and SAT2, respectively. In addition, a 100% DSe of serotypes O- and SAT2-specific mAb-bELISAs was observed for porcine sera when the cut-off was 30%. All mAb-bELISAs developed in this study displayed high repeatability/reproducibility without cross-reactivity. Finally, the diagnostic performance of mAb-bELISAs was found to be better than or equivalent to compared with pAb-cELISAs, suggesting that mAb-bELISAs can be used to replace existing pAb-ELISAs for the detection of antibodies against these four FMDV serotypes.

Direct competitive immunoassay method for sensitive detection of the histamine in foods based on a MI-Cu-GMP nanozyme marker and molecularly imprinted biomimetic antibody.

Histamine may lead to low blood pressure, skin flushing and edema when it accumulates in large amounts in the body. Therefore, establishing sensitive methods for the detection of histamine in foods is extremely important to ensure food safety and human health. The MI-Cu-GMP NPs (2-methylimidazole-copper-guanosine monophosphate nanozymes) with high laccase-like activity were synthesized. Using the prepared molecular imprinted membrane as biomimetic antibody and MI-Cu-GMP NPs as marker, a sensitive direct competitive biomimetic enzyme-linked immunoassay (BELISA) method for rapid detection of the histamine in foods was developed. Under optimal conditions, the limit of detection (LOD, IC15) and sensitivity (IC50) of the BELISA method for histamine was 0.05 mg L-1 and 1.22 mg L-1, respectively. The liquor samples spiked with histamine was detected by the BELISA method with satisfactory recoveries ranging from 90.00% to 116.00%. Further, the level of histamine in three samples (cooking wine, rice vinegar and soy sauce) was tested by the BELISA and high-performance liquid chromatography (HPLC), with no significant difference found between the two methods. Given the advantages, the established BELISA method is expected to provide practical guidance for the monitoring of histamine in food and provides a foundation for the detection of other food hazards. © 2024 Society of Chemical Industry.

Prevalence of bluetongue virus disease in a small ruminant population in Kalat, Balochistan, Pakistan

Background and Aim: Bluetongue is a vector-borne, emerging disease that poses a severe threat to most domesticated animals. A cross-sectional study was conducted to estimate the prevalence of bluetongue virus (BTV) disease in apparently healthy sheep and goats in Kalat, Balochistan. Materials and Methods: A total of 270 serum samples (sheep: 150 and goat: 120) were collected and screened for the detection of anti-BTV antibodies using a competitive enzyme-linked immunosorbent assay. The data regarding different contributory risk factors were also collected using a predesigned questionnaire. Results: It revealed that overall, 27.4% (74/270, 95% confidence interval, χ2 = 1.71, p = 0.12) prevalence in both sheep and goat populations. The highest prevalence of 47% (32/68) was recorded in Surab city with the lowest prevalence of 15.49% (11/71) in the Manguchar area. In contrast, in Kalat 28.1% (9/32), Daan area 24% (12/50), and Marap area 22.44% (11/49), seropositivity was recorded. Upon sex bases, antibodies were almost equally found in both male 28.57% (8/28) and female 27.27% (66/242) animal populations. Moreover, all four breeds (Balochi, Khurasani, Lehri, and Rakhshani) were equally and potentially seropositive. The Khurasani breed was the most susceptible to 34.69% (17/49), followed by the Balochi breed, 45/145 (31%) seropositivity. The prevalence of BTV was 16.66% (1/6) in Rakhshani breed and 15.71% (11/70) in Lehri breed., Ticks were found in almost 21% of animals, while 93% of animals were reared on open grazing in rangelands. Conclusion: This study clearly indicates widespread BTV infection in small ruminants in the study area that may pose serious threats to livestock farming. Further extensive studies are recommended to study the prevalence of disease in different agroecological zones of the province. This also warns the high-ups to manage concrete efforts to eradicate and control the disease in the area. Keywords: antibodies, Balochistan, bluetongue virus, competitive enzyme-linked immunosorbent assay, Kalat.

Pseudomonas aeruginosa exotoxin A as a novel allergen induced Non-TH2 inflammation in a murine model of steroid-insensitive asthma

BackgroundDespite the immediate in vivo occurrence of anaphylactic and allergic reactions following treatment with Pseudomonas aeruginosa exotoxin A (PEA)-based immunotoxins, the immunological role of PEA in asthma pathogenesis remains unclear. ObjectiveThis study investigated the allergenic potential of PEA and the specific type of asthma induced. MethodsRecombinant PEA (rPEA) lacking domain Ia (to eliminate non-specific cytotoxicity) was expressed, purified, and employed to detect serum PEA-specific IgE levels in asthmatic patients. Competitive ELISA assays were used to assess rPEA's IgE binding capacity and allergenicity. Additionally, rPEA-challenged C57BL/6 mice were subjected to inflammatory endotyping and therapeutic assays to characterize the allergic nature of PEA. ResultsPEA-specific IgE was identified in 17 (14.2 %) of 120 asthma patients. The rPEA-sensitized and challenged mice had increased PEA-specific immunoglobulins (such as IgE, IgG1 and IgG2a) and developed asthma-like phenotypes with airway hyperresponsiveness, severe airway inflammation, and airway remodeling. Lungs from these mice displayed significant increases in neutrophils and IL-17A+ cells. Innate lymphoid cells (ILCs) produced type 2 cytokines (IL-4, IL-5, and IL-13), whereas Th cells did not. Nonetheless, airway inflammation, rather than hyperresponsiveness, was elicited in non-sensitized mice upon challenge with rPEA. Importantly, rPEA-induced asthmatic mice were unresponsive to dexamethasone treatment. ConclusionPEA is a novel allergen that sensitizes asthmatic patients. Furthermore, mice developed steroid-resistant asthma, characterized by an atypical cytokine profile associated with non-TH2 inflammation, only after being sensitized and challenged with rPEA. These findings suggest a potentially significant role for PEA in asthma development, warranting consideration in clinical diagnosis and treatment strategies.

Stability in human serum and plasma of the HIV peptide drug candidate CIGB-210 and improved variants.

The peptide CIGB-210 inhibits HIV replication, inducing a rearrangement of vimentin intermediate filaments. The assessment of the in vitro serum and plasma stability of this peptide is important to develop an optimal pharmacological formulation. A half-life of 17.68 ± 0.59min was calculated for CIGB-210 in human serum by reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Eight metabolites of CIGB-210 were identified with this methodology, all of them lacking the N-terminal moiety. A previously developed CIGB-210 in-house competitive ELISA was used to compare the stability of CIGB-210 derivatives containing either D-amino acids, acetylation at the N-terminus, or both modifications. The half-life of CIGB-210 in serum was five times higher when measured by ELISA than by HPLC/MS, and twice higher in plasma as compared to serum. The substitution of D-asparagine on position 6 doubled the half-life, while D-amino acids on positions 8 and 9 did not improve the stability. The acetylation of the N-terminus resulted in a 24-fold more stable peptide in plasma. The positive effect of N-terminal acetylation on CIGB-210 serum stability was confirmed by the HPLC/MS method, as the half-life of the peptide was not reached after 2h of incubation, which represents more than a 6.8-fold increase in the half-life with respect to the original peptide.

Development and application of a competitive ELISA for the detection of antibodies against Salmonella Abortusequi in equids

Development and application of a competitive ELISA for the detection of antibodies against <i>Salmonella</i> Abortusequi in equids

Development of an immunochromatographic strip for the rapid detection of 11-dehydro-thromboxane B2 in human urine

Urinary 11-dehydro-thromboxane B2 (11-DH-TXB2) is a diagnostic marker for evaluating the effect of aspirin treatment in patients with cardiovascular disease. Therefore, it is of great clinical significance to develop a rapid detection method for 11-DH-TXB2 in urine. In this study, we prepared an ultrasensitive monoclonal antibody (mAb) against 11-DH-TXB2. The mAb belongs to the IgG1 subtype with kappa light chains. In an indirect competitive ELISA, it had a half-maximal inhibitory concentration of 3.63 ng/mL and a high affinity of 5.60 × 109 L/mol for 11-DH-TXB2. We then developed an immunochromatographic strip for point-of-care testing (POCT) of 11-DH-TXB2 in human urine. The strip provides a cut-off value and limit of detection of 500 ng/mL and 31.2 ng/mL, respectively. The result is obtained within 15 min. In summary, the proposed strip is a portable POCT tool for monitoring 11-DH-TXB2 in human urine.

Study of the Prevalence of Bovine Brucellosis (Brucella abortus) in General Proaño Parish: Evaluation using Rose Bengal Test and Confirmation through Competitive ELISA

Bovine brucellosis is an infectious pathology that compromises animal well-being and causes significant economic losses for livestock farmers. This disease leads to abortions and is caused by the bacterium Brucella abortus. In this research, the prevalence of bovine brucellosis (Brucella abortus) was estimated in General Proaño Parish using the Rose Bengal test and confirmation through competitive ELISA. Methodologically, The research was carried out between February and August 2023 on 100 animals from 25 locations in the Proaño parish, in the Morona canton, Ecuador. Blood serum was obtained from five breeds of cattle: Charolais, Holstein, Brown Swiss, Mestiza and Jersey. The collected samples were transported to the laboratory for their respective analysis. Five milliliters of venous blood were collected from the coccygeal vein, from which 1 mL of blood serum was obtained. The serum was used for disease diagnosis through the Rose Bengal test, and those that tested seropositive were reconfirmed using the competitive ELISA immunoassay method. The clinical analyses determined that there is a 0% prevalence of the pathology; based on these results, we can state that the null prevalence of Brucella may be due to environmental and geographical factors that influence its presence and transmission. Furthermore, the precision and sensitivity of the diagnostic methods used are crucial; however, the ELISA and Rose Bengal methods may have been insensitive and did not detect mild infections. Finally, Brucella infection may not be present during sampling due to temporal fluctuations in its prevalence. In conclusion, there is no scientific evidence of bovine brucellosis as the primary cause of abortions and gestational losses in the study area.

Development of a competitive ELISA based on the LSDV A33 antigen

Goatpoxvirus (GTPV), sheeppoxvius (SPPV), and the Lumpy skin disease virus (LSDV) is a Capripoxvirus belonging to the family poxviridae. They can cause significant economic losses in countries where this disease are endemic. However, effective and convenient diagnostic tools against sera antibody are not readily available until now. Toward this goal, a polyclonal antibody competitive enzyme-linked immunosorbent assay (c-ELISA) of detecting serogroup-specific antibody is established based on major LSDV antigen A33. Serum samples (n = 605) were collected to optimize the c-ELISA from different areas. The cut-off value for the c-ELISA was estimate using percent inhibition (PI) values. The diagnostic performance of test including sensitivity (sn) and specificity (sp) were obtained by receiver operator characteristic (ROC) analysis. Among these analysis, > 57.61% PI value was accepted as cut-off of the c-ELISA, the diagnostic sn an diagnostic sp were reached to 96.4% and 98.5%, at > 95% confidence interval. These results show that the developed competitive ELISA is sensitive, specific, and reliable, which make it appropriate for serological investigation.

Neopterin production in relation to COVID-19 in the Haut-Ogooué Province, Gabon

BackgroundIn sub-Saharan Africa, understanding of the immune process associated with the COVID-19 pandemic remains scarce. This study aimed to investigate the relationship between plasma neopterin concentrations and COVID-19 infection, focusing on changes over time and age-related changes in immune response.MethodsA retrospective case study was conducted during the first wave of COVID-19 from March to August 2020. Whole blood and associated symptoms and comorbidities were collected from patients of all ages and sexes. Concentrations of plasma neopterin were measured using a commercial competitive neopterin ELISA (Neopterin ELISA, IBL International GmbH, Germany).ResultsWe analyzed data for 325 patients: 38% (n = 124) with COVID-19, and 62% (n = 201) without COVID-19, as a control group. We found that plasma neopterin concentrations were significantly higher in the COVID-19 group (mean value 45.1 nmol/L (SD 19)) than in the control group (mean value 33.8 nmol/L (SD 13)) (p = 0.004). In addition, neopterin levels decreased gradually over time in patients with COVID-19 (p < 0.001). Moreover, ROC analysis found that the best cut-off value for diagnosing COVID-19 patients based on plasma neopterin levels was 38.85 nmol/L with 70% sensitivity and 82% specificity (AUC, 0.74 [0.69–0.82], p < 0.05). We also found an increase in neopterin production with increasing age (p < 0.001).ConclusionOur findings contribute to our growing understanding of neopterin levels as a promising biomarker for the detection of COVID-19 cases in sub-Saharan Africa.

Genome characterization of Rift Valley fever virus isolated from cattle, goats and sheep during interepidemic periods in Kenya

Rift Valley fever virus (RVFV) is a mosquito-borne RNA virus of the Phlebovirus genus in the phenuviridae family. Its genome is trisegmented with small (S), medium (M) and large (L) fragments. In nature, the virus exists as a single serotype that is responsible for outbreaks of Rift Valley fever (RVF), a zoonotic disease that often occurs in Africa and the Middle East. RVFV genomes are thought to undergo both recombination and reassortment and investigations of these events is important for monitoring the emergence of virulent strains and understanding the evolutionary characteristics of this virus. The aim of this study was to characterize the genomes of RVFV isolates from cattle, sheep, and goats collected during an interepidemic period in Kenya between June 2016 and November 2021. A total of 691 serum samples from cattle (n = 144), goats (n = 185) and sheep (n = 362) were analysed at the Central Veterinary Laboratories. The competitive IgM-capture ELISA, was used to screen the samples; 205 samples (29.67%) tested positive for RVFV. Of the 205 positive samples, 42 (20.5%) were from cattle, 57 (27.8%) from goats, and 106 (51.7%) from sheep. All the IgM-positive samples were further analyzed by qPCR, and 24 (11.71%) tested positive with Ct values ranging from 14.788 to 38.286. Two samples, 201808HABDVS from sheep and 201810CML3DVS from cattle, had Ct values of less than 20.0 and yielded whole genome sequences with 96.8 and 96.4 coverage, respectively. There was no statistically significant evidence of recombination in any of the three segments and also phylogenetic analysis showed no evidence of reassortment in the two isolated RVFV segments when compared with other isolates of different lineages from previous outbreaks whose genomes are deposited in the GenBank. No evidence of reassortment leaves room for other factors to be the most probable contributors of change in virulence, pathogenicity and emergence of highly virulent strains of the RVFV.

Longitudinal seroepidemiological survey and risk factors for bluetongue virus infection in sheep in the state of Parana, Brazil, from 2014 to 2017.

The Bluetongue disease is an infectious and non-contagious viral disease mainly transmitted through hematophagous vector of the Culicoides genus, to domestic and wild ruminants. The aim of this study was to evaluate the antibodies occurrence, persistence and potential risk factors associated with bluetongue virus infection in sheep flocks in the state of Parana, Brazil. The competitive ELISA test was applied to evaluate 690 blood serum samples from 22 farms in eight mesoregions of Parana in 2014, and 270 sheep blood serum samples from 10 of the 22 previously studied farms in 2017. A questionnaire was applied to evaluate the risk factors associated with BTV infection. In 2014 and 2017, the numbers of seroreactive sheep were found to be 28.26% (195/690) and 41.11% (111/270), respectively, representing 95.45% (21/22) and 90% (9/10) of the flocks. The significant variables considered as risk factors were Culicoides presence (P < 0.0001; OR = 8.83 and 95% CI 4.28-18.22); genealogical record (P < 0.0001; OR = 0.23 and 95% CI 0.12-0.45) and use of sheepfold (P = 0.0208; OR = 0.36 and 95% CI 0.15-0 0.86). It was determined that BTV infection is endemic in Parana and persists in the mesoregions where the climate is favorable to vector proliferation.

Interleukin-17A (IL-17A) is involved in antibody specificity to conformational epitopes

The specificity of antibodies (Ab) is essential for the precise recognition of foreign or dangerous molecules. We have shown that mice infected with non-pathogenic Lactate Dehydrogenase Elevating Virus (LDV) inoculated with human growth hormone (hGH) or Ovalbumin (OVA), exhibit modified specificity of anti-hGH or anti-OVA Ab associated with the secretion of IFN-γ, IL-13, and IL-17.Cytokines are directly or indirectly involved in the isotypes, specificity, and affinity of Ab. Accordingly, here we investigated the effect of IL-17 neutralization on Ab specificities to OVA and Diphtheria Toxoid (DTx) in a mouse model of viral infection. Thereby, we employed an anti-cytokine “auto-vaccination” with an OVA/IL-17A complex or a Monoclonal Ab (MAb) anti-IL-17A (MM17/F3).Competitive ELISA assays were used to estimate the quality of the humoral immune response and the amount of Abs to conformational versus linear antigenic determinants. Results indicated that the OVA/IL-17A complex increased Abs levels to conformational epitopes of OVA, while LDV prolonged antibodies for a longer period. Mice treated with MM17F3 MAb showed an increase in Abs to conformational epitopes of OVA. A similar effect, confirmed by a competitive Western-blot assay, was produced by LDV. Moreover, an increased level of IgM, IgG1, and IgG2a was found in infected animals. Similarly, MAb anti-IL-17A treatment increased the proportion of Ab to conformational epitopes of DTx in uninfected mice, while LDV decreased this parameter.In conclusion, our findings demonstrate a correlation between IL-17A neutralization and a change in Ab specificity to OVA or DTx, presenting a novel strategy for obtaining Abs with higher specificity.

Seroprevalence of PPR in Goats of Buffer Zone of Chitwan National Park

A study conducted from September to December 2019 aimed to assess the seroprevalence of Peste des Petits Ruminants (PPR) in goats residing in the buffer zone of Chitwan National Park. A total of 221 serum samples were collected from unvaccinated goat flocks across the buffer zone regions (48 from Bhojad, 64 from Bhandara, 66 from Padampur, 25 from Gondrang, and 18 from Kathar) using random sampling techniques. Competitive Enzyme-Linked Immunosorbent Assay (ELISA) was employed to detect antibodies against the PPR virus (PPRV). The study revealed an overall seroprevalence rate of 28.96%. The highest seroprevalence rate of 39.58% was observed in goats from Bhojad, while the lowest rate of 15.63% was found in goats from Bhandara. The findings emphasize the need for regular vaccination of small ruminants in Bhojad, Padampur, and Kathar to enhance immunity and prevent PPR infection. Additionally, such measures can help mitigate the transmission of PPR between goats and wild animals as they share common grazing lands.

Development and evaluation of an immunoassay for the quantification of N-acetylneuraminic acid (Neu5Ac) in foods and biosamples

N-acetylneuraminic acid is an active ingredient in tonic foods and an important additive in foods and biopharmaceuticals. To address the limitations of existing methods of N-acetylneuraminic acid quantification, we developed an immunoassay based on antibodies induced in hens using artificial antigen, showing high sensitivity and specificity with no cross-reactivity with eight N-acetylneuraminic acid analogues. An IgY-based indirect competitive enzyme-linked immunosorbent assay showed a detection range of 1.14 to 70.08 ng/mL and a limit of detection of 0.57 ng/mL. In spiked samples, recoveries by the indirect competitive enzyme-linked immunosorbent assay ranged from 74.05% to 110.87% compared with HPLC (73.01% to 108.8%). Consistency between the indirect competitive enzyme-linked immunosorbent assay and HPLC was satisfactory (R2 = 0.9736), demonstrating this established immunoassay as a rapid and reliable approach for N-acetylneuraminic acid analysis. The assay described in this study provides an important method for the screening of N-acetylneuraminic acid in biological samples and foodstuffs.