Antibody Enzyme-linked Immunosorbent Assay Research Articles

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18–25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening.Key points• QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies• Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits• QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus

Clinicomycological Study of the Spectrum of Pulmonary Aspergillosis at a Tertiary Care Hospital in Central India.

Knowing the spectrum, prevalence, and modes of diagnosis of pulmonary aspergillosis (PA) will be beneficial to clinicians for its early diagnosis and management. This study aims to estimate the prevalence, spectrum, and role of serological tests and radiological findings in the diagnosis of PA. A total of 150 patients were suspected of having PA after obtaining relevant clinical history and radiological imaging. The patients were grouped into each spectrum of PA as invasive PA (IPA), chronic necrotizing PA (CNPA), aspergilloma, allergic bronchopulmonary aspergillosis (ABPA) based on predisposing factors, clinical and radiological findings, and the guidelines of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG). Samples (bronchoalveolar lavage (BAL), sputum, blood) were collected from these patients and processed in a microbiology lab. BAL and sputum were subjected to microscopy by potassium hydroxide mount, calcofluor white mount, and culture. The serum was separated from blood by centrifugation and subjected to specific serological tests based on the spectrum of PA that the patient was suspected to have. For IPA, serum and BAL galactomannan antigen enzyme-linked immunosorbent assay (ELISA) was performed. For CNPA and aspergilloma, the anti-Aspergillus IgG antibody ELISA was performed. For ABPA, the tests performed were total immunoglobulin E (IgE) ELISA, Aspergillus fumigatus-specific IgE ELISA, and anti-Aspergillus immunoglobulin G (IgG) antibody ELISA. After compiling the clinical, radiological, culture, and serological findings, patients were diagnosed to have a particular spectrum of PA. The prevalence of IPA was 1.4%, CNPA was 4%, ABPA was 3.2%, and aspergilloma was 2.9%. CNPA was the predominant spectrum of PA in our study. Culture positivity for Aspergillus species was seen the highest in aspergilloma patients, followed by IPA, ABPA, and CNPA patients. A. fumigatus was the most common causative agent of PA, except for IPA for which Aspergillus flavus was the most common causative. Aspergillus niger and Aspergillus terreus were less the frequent causes of PA. A combination of radiological, microbiological, and serological tests along with clinical correlation is needed to confirm the diagnosis of PA.

Anti-HBsAg IgY polyclonal antibodies potential as capture antibody for HBsAg Detection Kit development

Hepatitis B, affecting about 296 million globally, is a significant concern, with Indonesia ranking second in Southeast Asia for case numbers. The disease's latent initial phase, devoid of early symptoms upon hepatitis B virus (HBV) infection, highlights the demand for precise diagnostics. This research aimed to develop anti-HBsAg polyclonal antibodies (pAb) for application as capture agents within a sandwich enzyme-linked immunosorbent assay (ELISA). Chicken egg-derived IgY antibodies have advantages over mammalian ones due to simpler extraction and higher yield. In a study involving 21-week-old chickens, four intramuscular injections of 500 µg HBsAg antigen in Freund's adjuvant were administered at two-week intervals. Eggs as IgY source were collected daily and then IgY was isolated from eggyolk using polyethylene glycol (PEG) precipitation. The Bradford method was used to measure the total protein concentration, while the existence of IgY and pAb specific IgY against Anti-HBsAg was verified through SDS-PAGE and sandwich ELISA using HRP as a reporter, respectively. The resulting SDS-PAGE showed two distinct IgY bands: a 68 kDa heavy chain and a 23 kDa light chain. Using these anti-HBsAg IgY antibodies as capturing agents, the slightly elevation of IgY pAb against HBsAg level has been identified within the second week following the initial immunization. Subsequently, from the third to the eighth week, antibody levels escalated significantly, ranging from 2 to 13-fold higher than those observed in the second week. These findings suggest the potential use of IgY pAb as effective capture antibodies in sandwich ELISA for HBsAg antigen detection.

Sustained Remission Without Corticosteroids Among Patients With Pemphigus Who Had Rituximab as First-Line Therapy

ImportanceThe Ritux 3 trial demonstrated the short-term efficacy and safety of first-line treatment with rituximab compared with a standard corticosteroid regimen in pemphigus. No data on the long-term follow-up of patients who received rituximab as first line are available.ObjectiveTo assess the long-term efficacy and safety of the Ritux 3 treatment regimen.Design, Setting, and ParticipantsThis 7-year follow-up study of the Ritux 3 trial included patients with pemphigus from 25 dermatology departments in France from January 1, 2010, to December 31, 2015.ExposurePatients were initially randomized in the rituximab plus prednisone group or prednisone-alone group.Main outcomes and measuresThe primary outcome was the 5- and 7-year disease-free survival (DFS) without corticosteroids, assessed by Kaplan-Meier curves. Secondary outcomes were occurrence of relapse, occurrence of severe adverse events (SAEs), and evolution of antidesmoglein (Dsg) antibody enzyme-linked immunosorbent assay values to predict long-term relapse.ResultsOf the 90 patients in the Ritux 3 trial, 83 were evaluated at the end of follow-up study visit (44 in the rituximab plus prednisone group; 39 in the prednisone-alone group) with a median (IQR) follow-up of 87.3 (79.1-97.5) months. Forty-three patients (93%) from the rituximab plus prednisone and 17 patients (39%) from the prednisone-alone group had achieved complete remission without corticosteroids at any time during the follow-up. Patients from the rituximab group had much longer 5- and 7-year DFS without corticosteroids than patients from the prednisone-alone group (76.7% and 72.1% vs 35.3% and 35.3%, respectively; P < .001), and had about half the relapses (42.2% vs 83.7%; P < .001). Patients who received rituximab as second-line treatment had shorter DFS than patients treated as first line (P = .007). Fewer SAEs were reported in the rituximab plus prednisone group compared with the prednisone-alone group, 31 vs 58 respectively, corresponding to 0.67 and 1.32 SAEs per patient, respectively (P = .003). The combination of anti-Dsg1 values of 20 or more IU/mL and/or anti-Dsg3 values of 48 or more IU/mL yielded 0.83 positive predictive value and 0.94 negative predictive value to predict long-term relapse.Conclusions and RelevanceIn this secondary analysis of the Ritux 3 trail, first-line treatment of patients with pemphigus with the Ritux 3 regimen was associated with long-term sustained complete remission without corticosteroid therapy without any additional maintenance infusion of rituximab.

Detection of Plasmodium knowlesi in whole blood samples with sandwich enzyme-linked immunosorbent assay (ELISA) using rhoptry-associated protein 1 specific polyclonal antibodies

ABSTRACT Background &amp; objectives: Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into new method developments to diagnose the disease. This study assessed the ability of the PkRAP-1 sandwich enzyme-linked immunosorbent (ELISA) to detect P. knowlesi antigens in whole blood specimens. Methods: Western blot assay was conducted to evaluate the ability of raised mouse and rabbit anti-PkRAP-1 polyclonal antibodies to bind to the native proteins in P. knowlesi lysate. The polyclonal antibodies were then used in sandwich ELISA to detect P. knowlesi. In the sandwich ELISA, mouse and rabbit polyclonal antibodies were used as the capture and detection antibodies, respectively. The limit of detection (LOD) of the assay was determined using P. knowlesi A1H1 culture and purified recombinant PkRAP-1. Results: Western blot results showed positive reactions towards the proteins in P. knowlesi lysate. The LOD of the assay from three technical replicates was 0.068% parasitaemia. The assay performance in detecting P. knowlesi was 83% sensitivity and 70% specificity with positive and negative predictive values of 74% and 80%, respectively. The anti-PkRAP-1 polyclonal antibodies did not cross-react with Plasmodium falciparum and healthy samples, but Plasmodium vivax by detecting all 12 samples. Interpretation &amp; conclusion: PkRAP-1 has the potential as a biomarker for the development of a new diagnostic tool for P. knowlesi detection. Further studies need to be conducted to establish the full potential of the usage of anti-PkRAP-1 antibodies for P. knowlesi detection.

Protective effects of a novel chimeric virus–like particle vaccine against virulent NDV and IBDV challenge

Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.

A third (booster) dose of the inactivated SARS-CoV-2 vaccine elicits immunogenicity and T follicular helper cell responses in people living with HIV.

This study sought to explore the immunogenicity of a booster dose of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in people living with human immunodeficiency virus (HIV) and identify the factors affecting the magnitude of anti-SARS-CoV-2 antibody levels. A total of 34 people living with HIV (PLWH) and 34 healthy donors (HD) were administered a booster dose of the same SARS-CoV-2 vaccine. Anti-SARS-CoV-2 antibody and immunoglobulin G (IgG) levels were measured using the SARS-CoV-2 S protein neutralizing antibody Enzyme-Linked Immunosorbent Assay (ELISA) and 2019-nCov IgG Chemiluminescent Immunoassay Microparticles, respectively. Spearman correlation analysis was used to measure the correlation between laboratory markers and neutralizing antibody and IgG levels. Peripheral blood mononuclear cells (PBMCs) were extracted from each subject using density gradient centrifugation and the numbers of memory T and T follicular helper (Tfh) cells were determined using flow cytometry. PLWH had a marked reduction in CD4 and B cell levels that was accompanied by a lower CD4/CD8 T cell ratio. However, those who received a supplementary dose of inactivated SARS-CoV-2 vaccines exhibited antibody positivity rates that were analogous to levels previously observed. The booster vaccine led to a reduction in IgG and neutralizing antibody levels and the amplitude of this decline was substantially higher in the PLWH than HD group. Correlation analyses revealed a strong correlation between neutralizing antibody levels and the count and proportion of CD4 cells. Anti-SARS-CoV-2 IgG antibody levels followed a similar trend. The expression of memory T and Tfh cells was considerably lower in the PLWH than in the HD group. PLWH had an attenuated immune response to a third (booster) administration of an inactivated SARS-CoV-2 vaccine, as shown by lower neutralizing antibody and IgG levels. This could be attributed to the reduced responsiveness of CD4 cells, particularly memory T and cTfh subsets. CD4 and cTfh cells may serve as pivotal markers of enduring and protective antibody levels. Vaccination dose recalibration may be critical for HIV-positive individuals, particularly those with a lower proportion of CD4 and Tfh cells.

The burden of the coronavirus disease 2019 virus infection in Burkina Faso: Results from a World Health Organization UNITY population-based, age-stratified sero-epidemiological investigation.

This study aimed to estimate the anti-SARS-CoV-2 antibody seroprevalence in the general population of Bobo-Dioulasso and Ouagadougou (Burkina Faso). We collected from March to April 2021 blood samples from randomly selected residents in both main cities based on the World Health Organization (WHO) sero-epidemiological investigations protocols and tested them with WANTAI SARS-CoV-2 total antibodies enzyme-linked immunosorbent assay (ELISA) kits intended for qualitative assessment. We also recorded participants' socio-demographic and clinical characteristics and information on exposure to SARS-CoV-2. Data were analysed with descriptive and comparative statistics. We tested 5240 blood samples collected between 03 March and 16 April 2021. The overall test-adjusted seroprevalence for SARS-CoV-2 antibodies was (67.8% [95% CI 65.9-70.2]) (N = 3553/3982). Seroprevalence was highest among participants aged 15-18 years old (74.2% [95% CI 70.5-77.5]) (N = 465/627), compared with those aged 10-14 years old (62.6% [95% CI 58.7-66.4]) (N = 395/631), or those over 18 (67.6% [95% CI 66.2-69.1]) (N = 2693/3982). Approximately 71.0% (601/860) of participants aged 10-18 years old who tested positive for SARS-CoV-2 antibodies experienced no clinical COVID-19 symptoms in the weeks before the survey, compared with 39.3% (1059/2693) among those aged over 18 years old. This study reports the results of the first known large serological survey in the general population of Burkina Faso. It shows high circulation of SARS-CoV-2 in the two cities and a high proportion of asymptomatic adolescents. Further studies are needed to identify the SARS-CoV-2 variants and to elucidate the factors protecting some infected individuals from developing clinical COVID-19.

Seroprevalence of Human Immunodeficiency Virus, Hepatitis C Virus, and Hepatitis B Virus Among Blood Donors in Sidi Bel Abbes, West Algeria.

Introduction Blood transfusions can transmit various viruses. Among them, the most common are hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). These viruses can cause fatal and life-threatening diseases. Worldwide, many people are infected with these viruses. Blood safety has made majorprogress in recent years. Yet, blood-borne viruses continue to be a major concern for patients, physicians, and policymakers. The aim of this study was to assessthe prevalence of HIV, HBV, and HCV in blood donors. Methods A cross-sectional study was performed to assess HIV, HBV, and HCV seropositivity in blood donors. This research was carried out at the blood bank of Sidi Bel Abbes University Hospital. This was a retrospective study based on records of blood donors. All data of 10,386 donors were analyzed from January 2015 to December 2015. Biological screening was performed by enzyme-linked immunosorbent assay (ELISA) using antibodies and/or antigens. The combined HCV and HIV antigen and antibody ELISA test was utilized. To confirm the results, the blood bank and the virology laboratory used the same technique in duplicate. Results The overall seroprevalence of blood-borne viral infections (HIV, HBV, and HCV) in blood donors was 0.8%. The prevalence of HIV was found to be 0.1%, while the prevalence of HBV and HCV was 0.4%. Coinfection was rare with only one case of HBV with HIV.There was a significant difference in seroprevalence rates among blood donors compared to the general population. Significant variations were observed between the prevalence of this study and those conducted in West, East, Central, and South African countries but not with those of neighboringNorth African countries. The study found no association between seropositivity in blood donors and factors like age, gender, donor status, type of donation, or site. Besides, HIV, HBV, or HCV prevalence was not influenced by ABO and Rhesus blood group. Conclusion The study showed that blood donors in Algeria have a lower prevalence of blood-borne viral infections than the general population. The seropositivity rate of viral markers was similar throughout North African countries. This rate remained low compared to other African countries. Residual risk of infection persists. There is a need to increase blood safety for recipients. This report is the first comprehensive overview of blood-borne viruses among Algerian blood donors. There is a need for further nationwide studies to get a whole picture of the situation.

Development of flavivirus subviral particles with low cross-reactivity by mutations of a distinct antigenic domain

The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections.Key points• The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs.• Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs.• Inoculation of mutated SVPs induces antibodies with low cross-reactivity.

Hepatitis E virus neutralization by porcine serum antibodies.

The consumption of raw or undercooked meat products poses a serious risk for human hepatitis E virus (HEV) infections. In many high-income countries, domestic pigs and wild boars represent the main animal reservoirs for HEV and are usually identified by reverse transcription-PCR and antibody enzyme-linked immunosorbent assay (ELISA). In order to characterize the humoral immune response in more detail, a cell culture-based serum neutralization assay using a culture-adapted HEV strain was established here. Measurement of neutralizing antibodies was only possible after removing the viral quasi-envelope by detergent treatment. Serum samples of 343 wild boars from Northern Germany were first analyzed for anti-HEV IgG using an in-house ELISA, resulting in 19% positive samples. Subsequently, a subset of 41 representative samples was tested with the neutralization assay, and the results correlated well with those obtained by ELISA. Not only the human HEV strain 47832c but also two porcine HEV strains were shown to be neutralized by porcine serum antibodies. Neutralizing activity was also found in samples containing both HEV-specific antibodies and HEV RNA. Testing of serum samples derived from two experimentally infected domestic pigs showed a steep increase in neutralizing activity at 24 or 51 days post infection, dependent on the used infectious dose. The developed assay can be useful for characterization of the humoral immune response after HEV infection and for assessing the efficiency of HEV vaccine candidates.

The seroprevalence of SARS-CoV-2 infection in Malaysia: 7 August to 11 October 2020.

From the beginning of the COVID-19 pandemic until mid-October 2020, Malaysia recorded ~15,000 confirmed cases. But there could be undiagnosed cases due mainly to asymptomatic infections. Seroprevalence studies can better quantify underlying infection from SARS-CoV-2 by identifying humoral antibodies against the virus. This study was the first to determine the prevalence of SARS-CoV-2 infection in Malaysia'sgeneral population, as well as the proportion of asymptomatic and undiagnosed infections. This cross-sectional seroprevalence study with a two-stage stratified random cluster sampling design included 5,131 representative community dwellers in Malaysia aged ≥1year. Data collection lasted from 7 August to 11 October 2020 involving venous blood sampling and interviews for history of COVID-19 symptoms and diagnosis. Previous SARS-CoV-2 infection was defined as screened positive using the Wantai SARS-CoV-2 Total Antibody enzyme-linked immunosorbent assay and confirmed positive using the GenScript SARS-CoV-2 surrogate Virus Neutralization Test. We performed a complex sampling design analysis, calculating sample weights considering probabilities of selection, non-response rate and post-stratification weight. The overall weighted prevalence of SARS-CoV-2 infection was 0.49% (95%CI 0.28-0.85) (N = 150,857). Among the estimated population with past infection, around 84.1% (95%CI 58.84-95.12) (N = 126 826) were asymptomatic, and 90.1% (95%CI 67.06-97.58) (N = 135 866) were undiagnosed. Our study revealed a low pre-variant and pre-vaccination seroprevalence of SARS-CoV-2 infection in Malaysia up to mid-October 2020, with a considerable proportion of asymptomatic and undiagnosed cases. This led to subsequent adoption of SARS-CoV-2 antigen rapid test kits to increase case detection rate and to reduce time to results and infection control measures.

Immunoassay with Novel Paired Antibodies for Detection of Lipoarabinomannan in the Pleural Fluid and Plasma of Patients with Tuberculous Pleurisy.

Tuberculous pleurisy (TP) is one of the most common forms of extrapulmonary tuberculosis, but its diagnosis is challenging. Lipoarabinomannan (LAM) antigen is a biomarker for Mycobacterium tuberculosis (Mtb) infection. LAM detection has potential as an auxiliary diagnostic method for TP. We have successfully generated five rabbit anti-LAM monoclonal antibodies (BJRbL01, BJRbL03, BJRbL20, BJRbL52, and BJRbL76). Here, anti-LAM antibodies were tested to detect LAM in the pleural fluid and plasma of patients with TP by sandwich enzyme-linked immunosorbent assays (ELISAs). The results revealed that all of the anti-LAM antibodies were successfully used as capture and detection antibodies in sandwich ELISAs. The BJRbL01/BJRbL01-Bio pair showed better performance than the other antibody pairs for detecting mycobacterial clinical isolates and had a limit of detection of 62.5 pg/mL for purified LAM. LAM levels were significantly higher in the pleural fluid and plasma of patients with TP than in those of patients with malignant pleural effusion or the plasma of non-TB, and LAM levels in the pleural fluid and plasma were positively correlated. Moreover, LAM levels in the pleural fluid sample were significantly higher in confirmed TP patients than in clinically diagnosed TP patients. Our studies provide novel LAM detection choices in the pleural fluid and plasma of TP patients and indicate that LAM detection assay has an auxiliary diagnostic value for TP, which may help to improve the diagnosis of TP.

Correlation of desmoglein 1 and 3 immunohistochemistry with autoantibody levels and clinical severity in pemphigus.

Pemphigus is a chronic potentially fatal autoimmune bullous disorder. Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are the two common subtypes. PV is the most common and aggressive type characterized by oral mucosal erosions and cutaneous lesions. PF presents with blisters on the scalp, face, and upper trunk, and spares the mucosae. Direct immunofluorescence (DIF) is the gold standard for diagnosis. Immunohistochemistry (IHC) is an emerging alternate diagnostic tool. In this study, our objectives were to identify the staining patterns of desmoglein 1 (dsg 1) and desmoglein 3 (dsg 3) IHC and to correlate the same with autoantibody levels and clinical severity in patients with PV and PF. Forty-nine clinically, histologically, and DIF-confirmed cases of pemphigus were included in the study. The IHC patterns were scored from 0 to 3+ with 3+ dsg 1 IHC exhibiting intense membranous staining in the upper layers of the epidermis and 3+ dsg 3 IHC showing intense basal layer staining. Enzyme-linked immunosorbent assay (ELISA) for anti-dsg 1 and 3 antibodies was performed in 38 cases where serum samples were available. The pemphigus disease activity index system was utilized for clinical scoring. A 0 to 1+ score was observed for dsg 1 IHC in 100% of PF cases. A score of 0 to 1+ was observed for dsg 3 IHC in 97.3% of PV cases. One hundred percent of cases with PF and 83.9% of patients with PV tested positive for ELISA anti-dsg 1 and 3 antibody titers, respectively. Anti-dsg 1 and 3 ELISA titers significantly correlated with the dsg 1 and dsg 3 IHC scores. The mucosal scores showed a significant association with both dsg 1 and 3 IHC (p < 0.001). The cutaneous scores showed a significant association with the dsg 3 IHC (p < 0.001). The IHC patterns for dsg 1 and 3 proved reliable in giving concordant results with the ELISA antibody titers and clinical severity.

A lateral flow immunochromatographic assay based on nanobody-oriented coupling strategy for aflatoxin B1 detection

Nonoriented coupling of nanobodies (Nb) to nanomaterials can considerably reduce epitope recognition or induce other dysfunctions, thereby restricting the application of Nb in field detection. Herein, using Avi-tag/streptavidin (Avi/SA)-oriented coupling strategy, we developed a lateral-flow immunochromatographic assay (LFIA) based on Nb-Avi/SA@quantum dots (QDs) probes (Nb-LFIA) for Aflatoxin B1 (AFB1) detection. Compared with nonoriented coupled Nb@QD probes, the detection sensitivity and stability of Nb-Avi/SA@QD probes were greatly retained. Furthermore, Nb showed higher tolerance to the extreme detection environments compared with monoclonal antibodies in traditional enzyme-linked immunosorbent assay and LFIA. After optimization, the limit of detection of Nb-LFIA was 0.095 ng mL−1, half-maximal inhibitory concentration was 0.85 ng mL−1, and the visual cut-off level was 1.25 ng mL−1. Nb-Avi/SA@gold nanoparticle probes were assessed for the wide applicability of the Avi/SA strategy, and they showed excellent performance (coefficient of determination = 0.999). Finally, the analysis performance of probes was assessed in oat samples, revealing a recovery rate of 88.8–116.7%, which was consistent with the results of high-pressure liquid chromatography coupled with tandem mass spectrometry. Altogether, our study provides a convenient and sensitive tool for AFB1 detection, and presents a viable path for efficient Nb application.

Development of a specific and sensitive sandwich enzyme-linked immunosorbent assay for the quantification of dasatinib

This study sought to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of dasatinib, a tyrosine kinase inhibitor. Anti-dasatinib antibodies were obtained from mice or rabbits by using two partial structures of dasatinib as haptens: 2-amino-N-(2-chloro-6-methylphenyl)-thiazole-5-carboxamide and 2-{4-(2-hydroxyethyl)-1-piperazinyl}-isonicotinic acid. The best combination of two antibodies for sandwich ELISA of dasatinib was determined using four anti-dasatinib antibodies derived from mice and rabbits. Using two dasatinib-specific rabbit antibodies, we successfully developed an ultra-specific and highly sensitive sandwich ELISA that is hardly affected by the main metabolite of dasatinib. The sandwich ELISA showed a linear detection range from 320 pg/mL to 1000 ng/mL. Serum dasatinib concentrations lower than 320 pg/mL were reproducibly measurable using the sandwich ELISA. The ELISA was specific to dasatinib and there were no cross-reactivities with the major metabolites 4′-hydroxy dasatinib and dasatinib carboxylic acid. The developed sandwich ELISA will be a valuable tool for pharmacokinetic studies of dasatinib. Furthermore, this study revealed that rabbit antibodies can sandwich drug molecules of a smaller size than mouse antibodies in sandwich ELISA.

SARS-CoV-2 Serological Investigation of White-Tailed Deer in Northeastern Ohio.

Coronaviruses are known to cross species barriers, and spill over among animals, from animals to humans, and vice versa. SARS-CoV-2 emerged in humans in late 2019. It is now known to infect numerous animal species, including companion animals and captive wildlife species. Experimental infections in other animals have established that many species are susceptible to infection, with new ones still being identified. We have developed an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to SARS-CoV-2 nucleocapsid (N) and spike (S) proteins, that is both sensitive and specific. It can detect S antibodies in sera at dilutions greater than 1:10,000, and does not cross-react with antibodies to the other coronaviruses tested. We used the S antibody ELISA to test serum samples collected from 472 deer from ten sites in northeastern Ohio between November 2020 and March 2021, when the SARS-CoV-2 pandemic was first peaking in humans in Ohio, USA. Antibodies to SARS-CoV-2 were found in serum samples from every site, with an overall positivity rate of 17.2%; we further compared the viral neutralizing antibody titers to our ELISA results. These findings demonstrate the need to establish surveillance programs to monitor deer and other susceptible wildlife species globally.

Associations between Johne’s disease and fertility in UK dairy herds

The objective of this observational study was to quantify associations between Mycobacterium avium subspecies paratuberculosis (MAP) antibody status and a variety of fertility outcomes, in UK dairy cattle. Longitudinal milk recording, fertility and MAP antibody enzyme-linked immunosorbent assay (ELISA) milk test data were collated retrospectively from 121,762 lactations in 78 herds. Datasets were structured into appropriate units to suit outcomes and enable temporal association between current and future MAP status, and fertility measures. Current MAP status was categorised according to most recent status within 180 days, with time-related future MAP status assigned based on MAP antibody ELISA milk test data for each cow. Multilevel multivariable logistic regression models were used to evaluate associations between MAP status and 21-day pregnancy and submission rate and conception risk. Posterior predictions and cross-validation techniques were used to assess model fit and check model building assumptions. A negative association was found between risk of insemination (Odds Ratio [OR], 0.78; 95% Credible Interval [CI], 0.66–0.92) and conception occurring (OR, 0.65; CI, 0.5–0.84) and transition from negative to non-negative MAP test status in the next 30–90 days. A positive association was observed between risk of insemination (OR, 1.34; CI, 1.16–1.52) and conception occurring (OR, 1.26; CI, 1.11–1.43) and transition from negative to non-negative MAP test status in the next 90–180 days. Current positive MAP test status was negatively and positively associated with insemination (OR, 0.59; CI, 0.49–0.70) and conception risk (OR, 1.12; CI, 0.96–1.30), respectively. Herd managers will have had access to test results, declaring cows with past recent or multiple positive MAP antibody ELISA results not to be bred, negatively influencing insemination risk. Overall, these results demonstrate the temporal association between a positive MAP antibody ELISA result and dairy cow fertility outcomes, with particular variability prior to a positive MAP antibody ELISA result.

Development of an Affordable ELISA Targeting the SARS-CoV-2 Nucleocapsid and Its Application to Samples from the Ongoing COVID-19 Epidemic in Ghana.

The true nature of the populationspread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations is often not fully known as most cases, particularly in Africa, are asymptomatic. Finding the true magnitude of SARS-CoV-2 spread is crucial to provide actionable data about the epidemiological progress of the disease for researchers and policymakers. This study developed and optimized an antibody enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid antigen expressed in-house using a simple bacterial expression system. Nucleocapsid protein from SARS-CoV-2 was expressed and purified from Escherichia coli. Plasma samples used for the assay development were obtained from GhanaianSARS-CoV-2 seropositive individuals during the pandemic, while seronegative controls were plasma samples collectedfrom blood donors before the coronavirus disease 2019 (COVID-19) pandemic. Another set of seronegative controls was collected during the COVID-19 pandemic. Antibody detection andlevels within the samples were validated using commercial kits and Luminex. Analyses were performed using GraphPad Prism, and the sensitivity, specificity and background cut-off were calculated. This low-cost ELISA (£0.96/test) assay has a high prediction of 98.9%, and sensitivity and specificity of 97% and 99%, respectively. The assay was subsequently used to screen plasma from SARS-CoV-2 RT-PCR-positive Ghanaians. The assay showed no significant difference in nucleocapsid antibody levels between symptomatic and asymptomatic, with an increase of the levels over time. This is in line with our previous publication. This study developed a low-cost and transferable assay that enables highly sensitive and specific detection of human anti-SARS-CoV-2 IgG antibodies. This assay can be modified to include additional antigens and used for continuous monitoring of sero-exposure to SARS-CoV-2 in West Africa.

Progressive and regressive infection with feline leukemia virus (FeLV) in cats in southern Brazil: Prevalence, risk factors associated, clinical and hematologic alterations.

Enzyme-linked immunosorbent assay (ELISA) for viral antigen is commonly used for the diagnosis of progressive feline leukemia virus (FeLV) infection but is not able to determine the true prevalence of infection when used as the sole test. Additional testing to detect proviral DNA will identify regressive (antigen negative) FeLV infections as well as progressive infections. Therefore, this study aimed to determine the prevalence of progressive and regressive FeLV infection, outcome-associated factors, and hematologic changes. A cross-sectional study was performed on 384 cats selected from routine hospital care. Blood samples were subjected to complete blood count, ELISA for FeLV antigen and FIV antibody, and nested PCR amplifying the U3- LTR region and gag gene, which are conserved in most exogenous FeLV. The prevalence of FeLV infection was 45.6% (CI95% 40.6-50.6%). The prevalence of progressive infection (FeLV+P) was 34.4% (CI95% 29.6-39.1%), that of regressive infection (FeLV+R) was 10.4% (CI95% 7.4-13.4%), for discordant but positive results 0.8% (CI95% 0.75-0.84%), for FeLV+P coinfected with FIV 2.6% (CI95% 1.2-4.0%), and FeLV+R coinfected with FIV 1.5% (CI95% 0.3-2.7%). Male cats were three times more likely to be in the FeLV+P group. Cats coinfected with FIV were 4.8 times more likely to belong to the FeLV+R group. In the FeLV+P group, the main clinical changes were lymphoma (38.5%), anemia (24.4%), leukemia (17.9%), concomitant infections (15.4%), and feline chronic gingivostomatitis - FCGS (3.8%). In the FeLV+R group, the main clinical signs were anemia (45.4%), leukemia (18.2%), concomitant infections (18.2%), lymphoma (9.1%), and FCGS (9.1%). Cats in the FeLV+P and FeLV+R groups showed mainly thrombocytopenia (56.6% and 38.2%), non-regenerative anemia (32.8% and 23.5%), and lymphopenia (33.6% and 20.6%). Hemoglobin concentration, packed cell volume (PCV), platelet count, lymphocytes, and eosinophils in the FeLV+P and FeLV+R groups had lower medians than the control group (FeLV/FIV-uninfected, healthy). Erythrocyte and eosinophil counts were statistically different among the three groups, with the medians of the FeLV+P and FeLV+R groups being lower than those of the control group. In addition, the median PCV and band neutrophil counts were higher in FeLV+P than in FeLV+R. Our results show a high prevalence of FeLV, different factors associated with the course of infection, and more frequent and severe hematologic changes in progressive infections compared with regressive infections.