Antibody Enzyme-linked Immunosorbent Assay Research Articles

HPV16/18 antibodies 16-years after single dose of bivalent HPV vaccination: Costa Rica HPV vaccine trial.

The Costa Rica HPV Vaccine Trial provided initial evidence that 1 dose of the bivalent human papillomavirus (HPV) vaccine induces stabilizing antibody levels that may provide extended protection against HPV-16/18 infections. We report antibody seropositivity and stability 11 to 16 years after vaccination. We invited a random subset of Costa Rica HPV Vaccine Trial participants (n = 398) who had received 3 doses and all women (n = 203) who had received 1 dose at 18 to 25 years of age to follow-up visits 11, 14, and 16 years after vaccination. We calculated HPV-16 and HPV-18 seropositivity and assessed change in enzyme-linked immunosorbent assay antibody levels 11 to 16 years after vaccination among 500 participants. By year 16, 99.4% (95% confidence interval [CI] = 96.8% to 100.0%) and 100.0% (95% CI = 98.9% to 100.0%) of 1-dose and 3-dose recipients, respectively, were HPV-16 seropositive and 98.8% (95% CI = 95.9% to 99.9%) and 100% (95% CI = 98.9% to 100.0%) of 1-dose and 3-dose recipients, respectively, were HPV-18 seropositive. Between years 11 and 16, women who had received 3 doses had a small but statistically significant decrease in the geometric mean concentration for HPV-16 of ‒12.4% (95% CI = ‒16.3% to ‒8.4%) and HPV-18 of ‒13.4% (95% CI = ‒17.2% to ‒9.4%). Among women who had received 1 dose, the decrease was statistically significant for HPV-16 at ‒8.9 (95% CI = ‒14.2% to ‒3.1%) but nonsignificant for HPV-18. Geometric mean concentration ratios of 3:1 dose (year 16) were 3.0 and 2.2 for HPV-16 and HPV-18, respectively. HPV-16/18 seropositivity remained exceedingly high 16 years after vaccination. Over 5 years, small declines in antibodies were observed. Women should have protection for at least 20 years and likely much longer at the observed rate of decline.

Efficacy Evaluation of COVID-19 Vaccines in Patients with Autoimmune Rheumatic Diseases in Mashhad, Iran

vaccination. However, since the majority of studies in the related literature are focused on the efficacy of messenger RNA in rheumatic patients, the present study aimed to assess the efficiency of the inactivated whole virus vaccines and viral vector COVID-19 vaccines in rheumatic diseases in Mashhad, Iran. Methods: This prospective cross-sectional study was conducted on ARDs patients who were referred to private clinics and rheumatologic clinics of Ghaem and Imam Reza Hospitals, Mashhad, Iran, during 2021-22. Anti-neutralizing antibodies have been considered to check antibodies of Sinopharm and Barkat vaccines, and an anti-spike antibody was considered to check Sputnik V and AstraZeneca vaccine antibodies. Humoral immunity was investigated using the anti-spike Enzyme-linked immunosorbent assay and anti-neutralizing antibodies. Results: The obtained results showed that humoral immunity after COVID-19 vaccination was observed in 73.9% of the patients with ARDs. However, humoral immunity was lower in ARDs patients who took tacrolimus and higher in patients with a history of COVID-19 infection (χ2=5.84, p=0.01). The infection after the second COVID-19 vaccination was lower in patients with humoral immunity (χ2=5.69, p=0.01). Conclusion: This study demonstrated that a homologous second dose of an inactivated whole viral vector SARS-CoV-2 vaccine was safe and provided a remarkable antibody response in ARDs patients.

Potential use of anti-thrombospondin-related apical merozoite protein (TRAMP) polyclonal antibodies in sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium knowlesi.

Plasmodium knowlesi, primarily a zoonotic malaria species is the most common malaria pathogen in the Southeast Asia especially in Malaysian Borneo, Malaysia. Due to morphological resemblance of P. knowlesi to other human Plasmodium, the sensitivity for microscopic detection of P. knowlesi, which is the gold standard, is compromised. Thus, efforts have been made in finding alternatives for the disease diagnosis. This study described the potential use of anti-PkTRAMP polyclonal antibodies in sandwich ELISA for P. knowlesi detection. Anti-PkTRAMP polyclonal antibodies raised from mice and rabbit were first evaluated for their binding capability towards native proteins in P. knowlesi lysates using Western blot. These mice and rabbit polyclonal antibodies were then used in the sandwich ELISA as capture and detection antibodies, respectively. P. knowlesi A1H1 culture was utilised to determine the limit of detection (LOD) of this assay. Its clinical performance was determined by testing with archived human malaria and uninfected samples. Western blot analysis affirmed the polyclonal antibodies reactivity to P. knowlesi. The LOD obtained from three replicated assays was at 0.015% parasitaemia. The assay has 76% sensitivity and 75% specificity for P. knowlesi. Its positive and negative predictive values were 76% and 75%, respectively. No cross reactivity with P. falciparum and healthy samples was observed, except for P. vivax where 10 out of 12 samples were detected. In conclusion, anti-PkTRAMP polyclonal antibodies can be useful in detecting P. knowlesi. Regardless, the full potential of anti-PkTRAMP antibodies for diagnostic purposes need to be explored further.

Hepatitis C virus genotypes among population with reported risk factors in Assam, north-east India: Emergence of genotype-8.

Background & objectives Hepatitis C virus (HCV) exhibits extensive genetic diversity in infected hosts. There are few published reports of HCV genotype (GT) distribution from the north-east Indian States lying close to the 'Golden Triangle' known for illicit drug trafficking. Real-time knowledge of HCVGT distribution is important for studies on epidemiologic aspects and virus evolution and for the development of new target-specific, direct-acting antiviral drugs. This study aims to examine the distribution of HCVGTs and their subtypes in different risk groups from Assam, north-east India. Methods It is a hospital-based cross-sectional study. Plasma samples reactive for anti-HCV antibody in enzyme-linked immunosorbent assay (ELISA) were subjected to viral load test and genotyping by real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) or characterization of non-structural protein NS5B region by nested PCR. Nucleotide sequences were subjected to phylogenetic analysis. Results The most common HCVGT detected was GT-3 (95.89%), followed by GT-1 (3.42%), GT-6xa (0.34%) and GT-8 (0.34%). The mean age of subjects was 30.24 yr, and males outnumbered females. The most commonly associated risk factor was injecting drug use (IDU) (74.31%), followed by tattooing and/or piercing (33.22%), transfusion of blood/blood products (10.27%), and haemodialysis (9.25%). Co-infection with human immunodeficiency virus (HIV) was found in 17.8 per cent, and with Hepatitis B virus (HBV) in 3.42 per cent of the cases. Interpretation & conclusions The detection of HCVGT-8 makes this the first report from Assam and the second from India as per the authors' knowledge. This may indicate strain's endemic nature in India. The increasing trend of HCV infection among young IDUs and HCV-HIV co-infection indicates the need for enhancing surveillance and intensified prevention efforts among young adults.

Application of Recombinant Monoclonal Antibodies from Transgenic Chicken Bioreactors in Enzyme-Linked Immunosorbent Assay.

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.

Seroprevalence of SARS-CoV-2 antibodies among children receiving primary care in Toronto, Ontario.

Characterizing the seroprevalence of SARS-CoV-2 antibodies in children is needed to optimize the COVID-19 public health response. We quantified the seroprevalence of SARS-CoV-2 infection-acquired antibodies and vaccine-acquired antibodies among children receiving primary care in Toronto, Canada. We conducted a longitudinal cohort study between January 2021 and November 2022 in healthy children aged 0-16years receiving primary care in Toronto. The primary and secondary outcomes were seroprevalence of SARS-COV-2 infection-acquired antibodies and vaccine-acquired antibodies ascertained from finger-prick dried blood spots. Samples were tested using an enzyme-linked immunosorbent assay for antibodies to full-length spike trimer and nucleocapsid. We explored sociodemographic differences with Firth's penalized generalized estimating equations. Of the 475 participants, 50.1% were girls and mean age was 6.4years (SD = 3.2). We identified 103 children seropositive for infection-acquired antibodies, with a crude seroprevalence that rose from 2.6% (95%CI 1.39-4.92) from January to July 2021 to 50.7% (95%CI 39.5-61.8) by July to November 2022. Seroprevalence of vaccine-acquired antibodies was 45.2% by July to November 2022 (95%CI 34.3-56.58). No differences in sociodemographic factors (age, sex, income, or ethnicity) were identified for infection-acquired antibodies; however, children with vaccine-acquired antibodies were more likely to be older, have mothers with university education, and have mothers who had also been vaccinated. Our results provide a benchmark for seroprevalence of SARS-CoV-2 antibodies in children in Toronto. Ongoing monitoring of the serological status of children is important, particularly with the emergence of new variants of concern, low vaccine coverage, and discontinuation of PCR testing.

Feline leishmaniosis in the Mediterranean Basin: a multicenter study

BackgroundCats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries.MethodsFrom 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities.ResultsOverall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis.ConclusionsThis study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin.Graphical

Possible pathogenic mechanisms for doxorubicin-induced splenic atrophy in a human breast cancer xenograft mouse model.

Doxorubicin-based chemotherapy is a widely used first-line treatment for breast cancer, yet it is associated with various side effects, including splenic atrophy. However, the pathogenic mechanisms underlying doxorubicin-induced atrophy of the spleen remain unclear. This study investigates that doxorubicin treatment leads to splenic atrophy through several interconnected pathways involving histological changes, an inflammatory response, and apoptosis. Immunohistochemical and western blot analyses revealed reduced size of white and red pulp, decreased cellularity, amyloidosis, and fibrotic remodeling in the spleen following doxorubicin treatment. Additionally, increased secretion of pro-inflammatory cytokines was detected using an antibody array and enzyme-linked immunosorbent assay (ELISA), which triggers inflammation through the regulation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-κB) signaling pathways. Further analysis revealed that the loss of regulators and effectors of the oxidative defense system, including sirtuin (Sirt)3, Sirt5, superoxide dismutase (SOD)1, and SOD2, was implicated in the upstream regulation of caspase-dependent cellular apoptosis. These findings provide insights on the pathogenic mechanisms underlying doxorubicin-induced splenic atrophy and suggest that further investigation may be warranted to explore strategies for managing potential side effects in breast cancer patients treated with doxorubicin.

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

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

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 developing new methods 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. 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. 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 P. falciparum and healthy samples, but P. vivax by detecting all 12 samples. 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.

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.

Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboardtitrationtest was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

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.

Enzyme-Linked Immunosorbent Assay for Antibodies Against the Tumor-Associated Antigen-Derived Cytotoxic T-Lymphocyte Epitope.

Antibodies serve as crucial indicators of the immune system in clinical tests. In therapeutic cancer vaccines, IgG antibodies against target antigens are vital for immune monitoring. Additionally, assessing baseline antigen-specific immune responses before cancer vaccine administration is possible by measuring IgM and IgG antibodies against the target antigen. To this end, we have developed an enzyme-linked immunosorbent assay (ELISA) system that detects and quantifies serum levels of IgG and IgM antibodies against the WT1 cytotoxic T-lymphocyte epitope peptide. The assay immobilizes the epitope peptide in a microplate to capture antigen-specific antibodies. Here, this article presents the details of our ELISA system to detect and measure antibodies against a tumor-associated antigen-derived cytotoxic T-lymphocyte epitope with high reproducibility. Detecting these antibodies has novel significance in the context of emerging critical roles of B lineage-cells in tumor immunity.

Are soluble E-selectin, ICAM-1, and VCAM-1 potential predictors for the development of diabetic retinopathy in young adults, 15-34 years of age? A Swedish prospective cohort study.

The aim of this study was to determine plasma levels of three adhesion molecules that may contribute to the development of diabetic retinopathy; soluble endothelial selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1), in young adults, aged 15-34 years at diagnosis of diabetes, to find potential predictors for development of retinopathy, and to evaluate their relation to diabetes associated autoantibodies. Participants with type 1 (n = 169) and type 2 diabetes (n = 83) were selected from the complications trial of the Diabetes Incidence Study in Sweden and classified in two subgroups according to presence (n = 80) or absence (n = 172) of retinopathy as determined by retinal photography at follow-up 8-10 years after diagnosis of diabetes. Blood samples were collected at diagnosis in 1987-88. The levels of sE-selectin, sICAM-1, and sVCAM-1 were analysed by enzyme-linked immunosorbent assay and islet cell antibodies by a prolonged two-colour immunofluorescent assay. Mean HbA1c (p<0.001) and clinical characteristics: mean body mass index (p = 0.019), systolic blood pressure (p = 0.002), diastolic blood pressure (p = 0.003), male gender (p = 0.026), and young age at diagnosis of diabetes (p = 0.015) remained associated with development of retinopathy in type 1 diabetes. However, in a multivariate analysis only HbA1c remained as a risk factor. sE-selectin was significantly higher in the group with type 2 diabetes and retinopathy, compared to the group with type 2 diabetes without retinopathy (p = 0.04). Regarding sE-selectin, sICAM-1, and sVCAM-1 in participants with type 1 diabetes, no differences were observed between the groups with or without retinopathy. This trial confirmed the role of HbA1c and clinical characteristics as predictors for development of retinopathy in type 1 diabetes. sE-selectin stands out as a potential predictor for development of retinopathy in type 2 diabetes, whereas a predictive role for sICAM-1 and sVCAM-1 could not be identified neither for type 1 nor type 2 diabetes.

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.