Enzyme-linked Immunosorbent Assay Reactivity Research Articles

Digital Quantification and Ultrasensitive Detection of Single Influenza Virus Using Microgel-in-Droplet Enzyme-Linked Immunosorbent Assay.

Detection and quantification of viral particles (VPs) facilitate both diagnostics of pathogenic viruses and quality control testing of virus-based products. However, existing technologies fail to afford concurrent ultrasensitive detection and large-scale absolute quantification of VPs. Here, we propose a digital Microgel-in-Droplet enzyme-linked immunosorbent assay (ELISA) system that enables the processing and monitoring of millions of ELISA reactions at the single-VP level by incorporating droplet microfluidics with sandwich ELISA. Upon validating the microfluidic workflow and optimizing ELISA parameters, we demonstrate ultrasensitive VP detection at a limit of detection of 56 PFU/test. Leveraging a fluorescence-based screening platform, we further realize high-throughput digital counting of VPs with a linear detection range of 500-64 000 PFU/test. The precision is comparable to that of the gold standard, the plaque assay, across a wide range of virus concentrations. We anticipate that our system will provide a novel paradigm for the absolute enumeration of various types of viral particles.

Üçüncü Basamak Bir Hastanenin Transfüzyon Merkezine Başvuran Donörlerde Sifiliz Tarama Sonuçlarının Değerlendirilmesi

Amaç: Kan bankacılığında Treponema pallidum'un neden olduğu sifiliz tanısı halk sağlığı açısından önemlidir. Donörler arasındaki olası vakaların yakalanması ve böylece donör kaybının en aza indirilmesi için en uygun algoritmanın benimsenmesi gerekmektedir. 2003 yılında Dünya Sağlık Örgütü ve 2010 yılında Avrupa Hastalık Önleme ve Kontrol Merkezi, sifiliz taramalarına Treponemal testlerle başlanmasını tavsiye etti. Bu çalışmada, kan bağışçısı adaylarında sifiliz taramasında reaktif Enzime Bağlı İmmünosorbent Test (ELISA) sonuçları ile yapılan doğrulayıcı testlerin sonuçlarının araştırılması amaçlandı.
 Gereç ve Yöntemler: Retrospektif olarak yapılan bu çalışmada, 2014-2021 yılları arasında Ankara Gülhane Eğitim ve Araştırma Hastanesinin kan bankasına kan bağışçısı olarak başvuran 69.127 bağışçı incelendi. Sifiliz tarama testinde reaktif çıkan donör adaylarından alınan örnekler, doğrulama testleri için Türkiye Halk Sağlığı Kurumu Mikrobiyoloji Referans Laboratuvarı'na gönderildi. Doğrulama testleri olarak Floresan Treponemal Antikor Emilimi (FTA-ABS) veya Treponema Pallidum Hemaglütinasyon Testi (TPHA) testleri kullanıldı.
 Bulgular: 128 donörde (%0,18) tekrar tekrar reaktif test sonuçları elde edildi. Doğrulayıcı bir test olarak 32 donöre TPHA uygulandı ve 11 pozitif sonuç (%34,3) elde edildi (Yanlış pozitiflik oranı = %65,7). FTA-ABS, doğrulama testi olarak 96 donörde yapıldı ve 59'unun (%61,4) testi pozitif çıktı (Yanlış pozitiflik oranı = %38,6). ELISA sinyal-kesme oranı beşin üzerinde olan 48 donörden 42'sinde FTA-ABS testi yapıldı ve 41 pozitif ve bir negatif sonuç elde edildi.
 Sonuçlar: Kan transfüzyonu ile ilişkili Treponema pallidum bulaşması önemli bir istenmeyen olay olduğundan, kan donörlerinin taranması için en uygun testlerin kullanılması gerekmektedir. Bu çalışma, FTA-ABS'nin ELISA-reaktif donörler için doğrulayıcı bir test olarak kullanılmasının, sifiliz tespit doğrulanmasını arttırdığını göstermektedir.

Silver nanoparticles enhanced enzyme-linked immunosorbent assay (ELISA) detection of cancer testis antigens (CTAs).

The Enzyme-Linked Immunosorbent Assay (ELISA) has been a cornerstone technique in laboratory medicine for over 55 years, relying on the specific binding of antibodies to antigens. ELISA's widespread use stems from its ability to detect low concentrations, its specificity, reproducibility, and potential for high-throughput screening. However, its sensitivity has limitations, prompting the exploration of innovative methods to improve the limit of detection (LOD). Nanoparticles provide a promising platform for enhancing ELISA sensitivity. Due to their high surface-to-volume ratio, they offer increased binding sites for capture elements and reporting tags, leading to amplified analytical signals. Recent studies have demonstrated improved sensitivity in ELISA through nanoparticle application, yielding faster detection times and enhanced sensitivities. This study investigates the potential of 50 nm citrate-capped silver nanoparticles to enhance ELISA's performance in quantifying cancer testis antigens (CTAs). In our study, we used the Human NY-ESO-1 ELISA kit (for research purposes) to determine the concentration of CTAs in randomly selected samples from healthy (n=89) and oncological (n=80) subjects, aged 18-75. We employed 50 nm citrate-capped silver nanoparticles (AGCB50-1M, BioPure Silver Nanoparticles - bare citrate, nano-Composix, San Diego, CA, USA). ELISA reactions followed the manufacturer's instructions, and data processing aligned with the same guidelines. Absorbance (OD) measurements occurred at 450 nm, influencing nanoparticle selection. Each ELISA well contained 5 ml of nanoparticles' stock solution with specified concentrations. CTAs concentrations were derived from the standard curve through CurveExpert Basic software. Statistical analysis was performed using SPSS v. 27 software, with p-values indicating significance if <0.03. The study adhered to Helsinki Declaration principles and received ethical approval. Participants provided informed written consent. The increased concentration values of CTAs for healthy individuals and cancer patients were determined in the case of the application of silver nanoparticles. The usage of nanoparticles can enhance the sensitivity of the ELISA method and positively influence its specific detection limit.

Evaluation of a mimotope of the Rickettsia outer membrane protein A (OmpA) as an antigen in enzyme-linked immunosorbent assay to detect rickettsiosis in capybaras (Hydrochoerus hydrochaeris), horses (Equus caballus), and opossums (Didelphis sp.).

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever, which is an important tick-borne zoonosis and, in Brazil, it causes Brazilian spotted fever, which has high lethality rate. This study aimed to evaluate a synthetic peptide corresponding to a segment of the outer membrane protein A (OmpA) as an antigen in a serological test for the diagnosis of rickettsial infections. The amino acid sequence of the peptide was selected by predicting B cell epitopes using B Cell Epitope Prediction (Immune Epitope Database and Analysis Resource) and Epitopia and OmpA sequences of Rickettsia rickettsii strain 'Brazil' and Rickettsia parkeri strains 'Maculatum 20' and 'Portsmouth'. A peptide with amino acid sequence common to both Rickettsia species was synthesized and arbitrarily named OmpA-pLMC. To evaluate this peptide in enzyme-linked immunosorbent assay (ELISA), serum samples of capybara (Hydrochoerus hydrochaeris), horse (Equus caballus), and opossum (Didelphis albiventris) that had been previously tested by indirect immunofluorescence assay (IFA) for rickettsial infection were separated into IFA-positive and IFA-negative groups and used in the assay. There were no significant differences in ELISA optical density (OD) values between IFA-positive and IFA-negative groups with horse samples. The mean OD values were significantly higher in the IFA-positive capybara serum samples (IFA-pos vs. IFA-neg = 2.389 ± 0.761 vs. 1.760 ± 0.840). However, receiver operating characteristic (ROC) curve analysis did not show significant diagnostic parameters. On the other hand, 12 out of 14 (85.7%) opossum samples of the IFA-positive group showed reactivity in ELISA, and this was significantly higher than of the IFA-negative group (0.7196 ± 0.440 vs. 0.2318 ± 0.098, respectively; 85.7% sensitivity, 100% specificity). Therefore, our results show that OmpA-pLMC has a potential to be used in immunodiagnostic assays to detect spotted fever group rickettsial infections.

Profiling Plasma Cytokines by A CRISPR-ELISA Assay for Early Detection of Lung Cancer

Cytokines play crucial roles in tumorigenesis and are potential biomarkers for cancer diagnosis. An Enzyme-linked Immunosorbent Assay (ELISA) is commonly used to measure cytokines but has a low sensitivity and can only detect a single target at a time. CRISPR-Associated Proteins (Cas) can ultra-sensitively and specifically detect nucleic acids and is revolutionizing molecular diagnostics. Here, we design a microplate-based CRISPR-ELISA assay to simultaneously profile multiple cytokines, in which antibodies are coupled with ssDNA to form antibody-ssDNA complexes that bridges CRISPR/Cas12a and ELISA reactions. The ssDNA triggers the Cas12a collateral cleavage activity and releases the fluorescent reporters to generate amplified fluorescent signals in the ELISA detection of cytokines. The CRISPR-ELISA assay can simultaneously measure multiple cytokines with a significantly higher sensitivity compared with conventional ELISA. Using the CRISPR-ELISA assay to profile plasma cytokines in 127 lung cancer patients and 125 cancer-free smokers, we develop a panel of plasma cytokine biomarkers (IL-6, IL-8, and IL-10) for early detection of the disease, with 80.6% sensitivity and 82.0% specificity. The CRISPR-ELISA assay may provide a new approach to the discovery of cytokine biomarkers for early lung cancer detection.

Resolvin D1 protects against Aspergillus fumigatus keratitis in diabetes by blocking the MAPK-NF-κB pathway

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

Comparative Analysis of BIOCHIP Mosaic-Based Indirect Immunofluorescence with Enzyme-Linked Immunosorbent Assay for Diagnosing Myasthenia Gravis.

Background: The detection of anti-acetylcholine receptor (AChR) and anti-muscle-specific tyrosine kinase (MuSK) antibodies is useful in myasthenia gravis (MG) diagnosis and management. BIOCHIP mosaic-based indirect immunofluorescence is a novel analytical method, which employs the simultaneous detection of anti-AChR and anti-MuSK antibodies in a single miniature incubation field. In this study, we compare, for the first time, the BIOCHIP MG mosaic with conventional enzyme-linked immunosorbent assay (ELISA) in the diagnosis of MG. Methods: A total of 71 patients with MG diagnosis were included in the study. Anti-AChR and anti-MuSK antibodies were measured separately by two different ELISA and simultaneously by BIOCHIP. The results were then compared. Results: The overall concordance between ELISA and BIOCHIP for anti-AChR reactivity was 74%. Cohen’s kappa was 0.51 (95% CI 0.32–0.71), which corresponds to 90% of the maximum possible kappa (0.57), given the observed marginal frequencies. The overall concordance for anti-MuSK reactivity was 84%. Cohen’s kappa was 0.11 (95% CI 0.00–0.36), which corresponds to 41% of the maximum possible kappa (0.27). Conclusion: The overall concordance among assays is not optimal.

Generation and Characterization of Nanobodies Against Tomato Leaf Curl Sudan Virus.

Begomoviruses infect food, fiber, and vegetable crop plants, including tomato, potato, bean, cotton, cucumber, and pumpkin, and damage many economically important crop plants worldwide. Tomato leaf curl Sudan virus (ToLCSDV) is the most widespread tomato-infecting begomovirus in Saudi Arabia. Using phage display technology, this study isolated two camel-derived nanobodies against purified ToLCSDV virions from a library of antigen-binding fragments (VHH or nanobody) of heavy-chain antibodies built from an immunized camel. The isolated nanobodies also cross-reacted with purified tomato yellow leaf curl virus virions and showed significant enzyme-linked immunosorbent assay reactivity with extracts from plants with typical begomovirus infection symptoms. The results can pave the way to developing diagnostics for begomovirus detection, design, and characterization of novel nanomaterials based on virus-like particles, in addition to nanobody-mediated begomovirus resistance in economically important crops, such as tomato, potato, and cucumber.

Composable Microfluidic Plates (cPlate): A Simple and Scalable Fluid Manipulation System for Multiplexed Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is the gold standard method for protein biomarkers. However, scaling up ELISA for multiplexed biomarker analysis is not a trivial task due to the lengthy procedures for fluid manipulation and high reagent/sample consumption. Herein, we present a highly scalable multiplexed ELISA that achieves a similar level of performance to commercial single-target ELISA kits as well as shorter assay time, less consumption, and simpler procedures. This ELISA is enabled by a novel microscale fluid manipulation method, composable microfluidic plates (cPlate), which are comprised of miniaturized 96-well plates and their corresponding channel plates. By assembling and disassembling the plates, all of the fluid manipulations for 96 independent ELISA reactions can be achieved simultaneously without any external fluid manipulation equipment. Simultaneous quantification of four protein biomarkers in serum samples is demonstrated with the cPlate system, achieving high sensitivity and specificity (∼ pg/mL), short assay time (∼1 h), low consumption (∼5 μL/well), high scalability, and ease of use. This platform is further applied to probe the levels of three protein biomarkers related to vascular dysfunction under pulmonary nanoparticle exposure in rat's plasma. Because of the low cost, portability, and instrument-free nature of the cPlate system, it will have great potential for multiplexed point-of-care testing in resource-limited regions.

Establishment and Application of a Method for the Determination of Ganoderic Acid A

A method for the quantitative determination of ganoderic acid A was constructed using the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA), and this method was used to determine the ganoderic A contents of Ganoderma lucidum samples in the market. The conjugate of ganoderic acid A and bovine serum albumin was used for four rounds of immunization on test rabbits to obtain rabbit antiganoderic acid A antibody IgG. The enzyme-labeled plate was coated with the conjugate of ganoderic acid A and ovalbumin. The first stage reaction in the indirect competitive ELISA was that the conjugate of ganoderic acid A in the sample competed with the conjugate coated on the enzyme-labeled plate to bind rabbit antibodies. The second stage reaction was the combination of goat anti-rabbit IgG–horseradish peroxidase and rabbit antiganoderic acid A antibody IgG. The results of the determination of ganoderic acid A standard by this method showed that the coefficient of variation of repeated wells in the group was &lt;5%, the detection limit of ganoderic acid A was 0.6 μg/L, and ganoderic acid A had a substantial dose-response relationship in the content range of 0.9–72.9 μg/L (R2 = 0.994). This method was used to measure the ganoderic A content of 12 varieties of G. lucidum in the market and showed the obvious differences in the ganoderic acid A contents of the different varieties. This method is simple, fast, and of great importance to the quality control of Ganoderma products.

Seroprevalence of SARS-CoV-2 infection among pet animals in Croatia and potential public health impact.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) emerged in late 2019 and has since caused a global pandemic. Experimental studies and sporadic reports have confirmed susceptibility of dogs and cats to SARS‐CoV‐2 infection. However, the importance of pet animals in the epidemiology of this infection is unclear. This study reports on a first large‐scale serosurvey of SARS‐CoV‐2 infections in dogs and cats in Europe. From 26 February 2020, just one day after the first confirmed human case of SARS‐CoV‐2 infection in Croatia, to 15 June 2020, dog and cat serum samples were collected from animals admitted to three veterinary facilities in Croatia. Additionally, on 25 May 2020, a total of 122 serum samples from employees of the Faculty of Veterinary Medicine University of Zagreb were collected. Total of 656 dogs and 131 cat serum samples were tested using an in‐house microneutralisation test (MNT). Human serum samples, as well as 172 randomly selected, dog sera were tested using enzyme‐linked immunosorbent assay (ELISA). ELISA‐positive human sera were subsequently tested using MNT. Neutralising antibodies were confirmed in 0.76% cats and 0.31% dogs. ELISA reactivity was recorded in 7.56% tested dog sera. On the other hand, 5.19% of administrative, basic and pre‐clinical sciences department personnel and 5.13% of animal health service providers and laboratory personnel tested ELISA positive. Neutralising antibodies were not confirmed in any of the human samples. In conclusion, seropositivity among pet animals in Croatia is low, especially when compared to results from China. A small number of seropositive animals with a low titre of neutralising antibodies suggest infections are rare and are following infections in the human population. Additionally, contact with animals does not seem to be an occupational risk for veterinary practitioners.

Comparative Evaluation and Measure of Accuracy of ELISAs, CLIAs, and ECLIAs for the Detection of HIV Infection among Blood Donors in China.

Background Enzyme-linked immunosorbent assay (ELISA) is the only serological method approved for blood screening in China. Automated chemiluminescence immunoassay (CLIA) and electrochemiluminescence immunoassay (ECLIA) had been used in clinical laboratories but not applied to screen HIV among blood donors. This study aimed to evaluate the performance of ELISA, CLIA, and ECLIA, focusing on the feasibility of CLIA/ECLIA for blood screening. Method 1029 blood donations from 14 blood centers screened by ELISA were enrolled in the study. All plasma samples were tested by eight ELISA assays in 16 blood centers, followed by the detection of CLIA and ECLIA methods in the National Center for Clinical Laboratories (NCCL), further confirmed by nucleic acid testing (NAT) and Western blot (WB). Results Of 1029 samples, 136 were confirmed as HIV positive. CLIA and ECLIA assay had similar sensitivities with ELISAs but showed higher specificity (CLIA: 99.1%, 885/893; ECLIA: 99.0%, 884/893), concordance rate (CLIA: 99.2%, 1021/1029; ECLIA: 99.1%, 1020/1029), and positive predictive value (PPV) (CLIA: 94.4%, 136/144; ECLIA: 93.8%, 136/145) than most of ELISA kits (>5 ELISAs) (P < 0.05). Kappa values of CLIA (0.967) and ECLIA (0.963) were the highest among all the serologic assays. Among 451 samples with initial ELISA reactivity, 315 were negatives, of which 307 (97.5%) and 306 (97.1%) were detected as nonreactive by CLIA (8 nonspecific reactions) and ECLIA (9 nonspecific reactions), respectively. Conclusion Compared with ELISA, CLIA and ECLIA are more specific and accurate in detecting HIV antibody/antigen and can keep more nonspecifically reactive donors detected by ELISA. CLIA and ECLIA can be used for the improvement of serological blood screening strategy to avoid the unnecessary loss of blood donors.

Elimination of the major allergen tropomyosin from shrimp muscle by boiling treatment

Tropomyosin (TM) is the major allergen found in invertebrates, including shrimp. To reduce the allergenicity of shrimp muscle, attempts were made to eliminate TM from the muscle by boiling. By boiling cubes (~ 1 g) of shelled shrimp muscle in 10 volumes of water (w/v) for 20 min, 7.1% of TM remained as determined by SDS-PAGE. Increasing the boiling water up to 2000 volumes of water did not result in improved elimination. When the muscle cubes were treated at 121 °C for 20 min in 2000 volumes of water, enzyme-linked immunosorbent assay (ELISA) reactivity against TM was reduced to 2.0 × 10−2 fold. After successive second and third treatments under the same conditions, ELISA reactivity was reduced to 4.2 × 10−5 and 1.6 × 10−5 folds, respectively. In addition, TM content in the muscle after the third treatment was lower than the threshold value for mandatory labeling (10 μg of ingredient proteins per 1 g of food). Thus, boiling treatment, especially high-pressure heating, can be an effective method for providing hypoallergenic shrimp for those who are allergic to shrimp and related invertebrates.

Development of monoclonal mouse antibodies that specifically recognize pancreatic polypeptide.

Pancreatic polypeptide (PP) is a 36-amino acid peptide encoded by the Ppy gene, which is produced by a small population of cells located in the periphery of the islets of Langerhans. Owing to the high amino acid sequence similarity among neuropeptide Y family members, antibodies against PP that are currently available are not convincingly specific to PP. Here we report the development of mouse monoclonal antibodies that specifically bind to PP. We generated Ppy knockout (Ppy-KO) mice in which the Ppy-coding region was replaced by Cre recombinase. The Ppy-KO mice were immunized with mouse PP peptide, and stable hybridoma cell lines producing anti-PP antibodies were isolated. Firstly, positive clones were selected in an enzyme-linked immunosorbent assay for reactivity with PP coupled to bovine serum albumin. During the screening, hybridoma clones producing antibodies that cross-react to the peptide YY (PYY) were excluded. In the second screening, hybridoma clones in which their culture media produce no signal in Ppy-KO islets but detect specific cells in the peripheral region of wild-type islets, were selected. Further studies demonstrated that the selected monoclonal antibody (23-2D3) specifically recognizes PP-producing cells, not only in mouse, but also in human and rat islets. The monoclonal antibodies with high binding specificity for PP developed in this study will be fundamental for future studies towards elucidating the expression profiles and the physiological roles of PP.

Trypanosoma cruzi infection in naturally infected dogs from an endemic region of Cundinamarca, Colombia.

The seropositivity and risk factors for Trypanosoma cruzi infection in dogs from a municipality of Cundinamarca, a central state of Colombia were studied. A total of 356 client-owned dogs from urban, peri-urban and rural areas of La Mesa municipality, (Cundinamarca, Colombia) were randomly selected. Blood samples were collected by venipuncture. Anti-T. cruzi antibodies were determined using the enzyme-linked immunosorbent assay (ELISA) method. Reactive ELISA sera were processed by indirect immunofluorescence to confirm the presence of anti-T. cruzi antibodies. Chi-square tests were conducted for statistical analysis. Serologic tests for T. cruzi infection showed a prevalence of 29.49% (105/356), the rural area show a highest T. cruzi infection pattern in comparison with the other zone locations. Two triatomine species were found through the study: Panstrongylus geniculatus (53.4%) and Rhodnius colombiensis (46.6%). The prevalence of positive vectors for parasite was of 52.1% (38/73). Additionally, a very close relation between triatomine bugs and dogs in the rural zone (1:3.1) was observed. These results are the first report of natural infection by T. cruzi in domestic dogs in La Mesa municipality. In conclusion, the presence of anti-T. cruzi antibodies in dogs in this area suggest vector transmission. There is a need for active surveillance programs throughout the La Mesa municipality and vector control strategies should also be implemented.

Performance, serologic and tracheal responses of laying hens exposed to F strain of Mycoplasma gallisepticum

ABSTRACT: The F strain of Mycoplasma gallisepticum (MG-F) protects chickens against mycoplasma infections, in which monitoring is made by serology and histopathology of trachea. This trial used 90 chickens, being 30 unvaccinated (G1 group), 30 eye-drop vaccinated at 8 weeks of age with MG-F (Ceva Animal Health, São Paulo, SP, Brazil) (G2), and 30 immunized at 8 and 11 weeks of age (G3). Samples were obtained from chickens on the 8, 12, 15, 18, 20 and 24th weeks of age for the enzyme-linked immunosorbent assay (ELISA) test. Tracheal fragments were collected after necropsies on the 15 and 24th weeks of age. Up to 12 weeks, the ELISA reactions in optical density (OD) were 0.165 (G1), 0.151 (G2) and 0.151(G3), all below 0.20 and with no significant difference among groups (p &gt; 0.05). After the 15th week, the ELISA reactions rose, yielding the following group averages by collecting dates: G1 (0.18, 0.19, 0.18, and 0.16), G2 (0.36, 0.49, 0.47, and 0.44) and G3 (0.41, 0.52, 0.59, 0.60), being the means in G2 and G3 not significantly different between than, but significantly different from G1. The initial weight (592.71, 621.33, and 594.40), the final weight (1,932.58, 1,987.59, and 1,875.20) and the weekly weight gain (11.65, 11.90, and 11.14) were not significantly different among groups. At necropsy the gross tracheal score means by group and dates were: 15th week (0.25, 0.61, and 0.54) and 24th week (0.54, 0.58, and 0.67), being these difference not significantly (p &gt; 0.05). On microscopy, the tracheal score averages by groups G1, G2 and G3, respectively, were: 15th week (0.25, 0.32, and 0.47) and 24th week (0.07, 0.75, and 0.08). G2 yielded higher score average than G1 and G3 on the 24th week. Higher tracheal changes for G2 and G3 as compared to G1 could be ascribed to MG-F infection. There were no evident prejudicial effects on live weight, weight gain and tissue changes by applying one or two vaccination doses.

Nominal effective immunoreaction volume of magnetic beads at single bead level.

Immunomagnetic bead (IMB)-based enzyme-linked immunosorbent assay (ELISA) has been the tool frequently used for protein detection in research and clinical laboratories. For most ELISA reactions the recommended dosage of IMBs is usually according to their weight (mg) or mass fraction (w/v) instead of the bead number. Consequently, the processes occurring in the immediate vicinity of the IMBs have always been ignored by researchers and they cannot be revealed in detail during the ELISA reaction. In this paper, we established the relationship between number of IMBs and colorimetric results, and further proposed a new concept of "nominal effective immunoreaction volume (NEIV)" to characterize a single IMB during ELISA reaction. Results showed that the NEIV of a single IMB has a constant value, which is unrelated to the amount of beads and the concentration of antigen. Optimal results of the colorimetric ELISA are achieved when the incubation volume meets each IMB's NEIV and is no longer enhanced by increasing the incubation volume. Thus, the reliable and relatively precise number of IMBs for ELISA detection during practical application could be determined. Most importantly, a study using IMB's NEIV would lay the foundation for a kinetics analysis of IMBs and antigens for future study.

Characterization of Antigenic Relatedness between GII.4 and GII.17 Noroviruses by Use of Serum Samples from Norovirus-Infected Patients.

A novel GII.17 norovirus variant caused major gastroenteritis epidemics in China in 2014 to 2016. To explore the host immune factors in selection of the emergence of this new variant, we characterized its antigenic relatedness with the GII.4 noroviruses that have dominated in China for decades. Through an enzyme-linked immunosorbent assay (ELISA) and a histo-blood group antigen (HBGA) blocking assay using sera from GII.4 and the GII.17 variant-infected patients, respectively, we observed limited cross-immune reactivity by the ELISA but little reactivity by the HBGA blocking assay between GII.4 norovirus and the new GII.17 variant. Our data suggest that, among other possible factors, GII.4-specific herd immunity had little role in the emergence of the new GII.17 variant. Thus, GII.17 may be an important active antigenic type or immunotype that needs to be considered for future vaccine strategies against human noroviruses.

Seropositivity for Trypanosoma cruzi in domestic dogs from Sonora, Mexico

BackgroundChagas disease is an important health problem in Latin America due to its incapacitating effects and associated mortality. Studies on seropositivity for Trypanosoma cruzi in Mexican dogs have demonstrated a direct correlation between seropositivity in humans and dogs, which can act as sentinels for the disease in this region. The objective of this study was to determine the seropositivity for T.cruzi infection in dogs from Sonora, a northern borderstate of Mexico.MethodsResponsible pet owners were selected at random from an urban area of Empalme municipality, Sonora, Mexico, and from there, 180 dog samples were collected. Anti-T. cruzi antibodies were determined using the enzyme-linked immunosorbent assay (ELISA) method. Reactive ELISA sera were processed by indirect immunofluorescence to confirm the presence of anti-T. cruzi antibodies. For the statistical analysis, chi-square tests were conducted.ResultsDogs’ sera showed a seropositivity rate of 4.44%. The rate of seropositivity was not associated with the dogs’ age, sex, or socioeconomics pertaining to the geographical area. One sample (1/180, 0.55%) showed the acute state of the disease.ConclusionsThe study found a presence of anti-T. cruzi antibodies in dogs in this area, which suggests vector transmission. There is a need for active surveillance programs throughout the state of Sonora and vector control strategies should also be implemented in endemic regions.

Immunodiagnostic potency of homologous antigens for natural Paramphistomum epiclitum infection in small ruminants in plate and paper enzyme linked immunosorbent assay

The objective of the present work was to standardize and evaluate indirect plate and dot- enzyme linked immunosorbent assay (ELISA) using purified Paramphistomum epiclitum homologous antigens in the small ruminants. Electrophoretic separation of somatic antigen (PeSAg) in reducing condition on 15% polyacrylamide gel resolved into 16 proteins of the molecular weight ranging from 14 -100 kDa. Two step ethanolic precipitation of supernatant of in-vitro culture of the fluke yielded P. epiclitum excretory-secretory antigen (PeESAg) of molecular weight 28 kDa. The animals (Goats=123; Sheep=91) were broadly kept into post-mortem and faecal examined groups. At many occasion the PeSAg found to cross reacts with other helminths parasites thus minimizing the specificity of the tests and antigens. There was no any direct correlation between the parasites load and ELISA reactivity pattern. The noted prevalence rate after combining the results of post-mortem examination and PeESAg based ELISA (plate and paper/ dot) was 30.08% (37/123) in goats and 28.57% (26/91) in sheep. While using PeESAg, the calculated overall sensitivity% was 92.86 (goats)/ 100 (sheep) in both plate and dot-ELISA, specificity% was 91.58 (goats)/ 91.55 (sheep) in plate ELISA while 88.42 (goats)/ 92.96 (sheep) in dot-ELISA, positive predictive value% was 76.47 (goats)/ 76.92 (sheep) in plate ELISA while 70.27 (goats)/ 80 (sheep) in dot-ELISA and negative predictive value% was 97.75 (goats)/ 100 (sheep) in plate ELISA while 97.67 (goats)/ 100 (sheep) in dot-ELISA, these values were optimum for the field sera sample so the tests and PeESAg can be recommended for the detection P. epiclitum infection in the small ruminants.