Detection Of Antibodies Research Articles

Development of a blocking ELISA employing a VP1-specific monoclonal antibody for the detection of DHAV3 antibodies.

Development of a blocking ELISA employing a VP1-specific monoclonal antibody for the detection of DHAV3 antibodies.

The prevalence and pattern of alloimmunization in patients with sickle cell disease in Abuja, Nigeria.

The prevalence and pattern of alloimmunization in patients with sickle cell disease in Abuja, Nigeria.

Multifunctional dopamine-modified conjugated polymer nanoparticles for ultrasensitive immunoassays.

Multifunctional dopamine-modified conjugated polymer nanoparticles for ultrasensitive immunoassays.

Low-background CRISPR/Cas12a sensing system with circular CRISPR RNA for amplified fluorescent detection of antibody in human serum.

Low-background CRISPR/Cas12a sensing system with circular CRISPR RNA for amplified fluorescent detection of antibody in human serum.

Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD): Insights into pathogenesis and biomarkers of prognosis.

Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD): Insights into pathogenesis and biomarkers of prognosis.

Fluorescent bead-based multiplex assays improve serological disease diagnostics and have potential of identifying sensitive immune biomarkers for maintaining health and performance.

Fluorescent bead-based multiplex assays (multiplex assays) for serological detection of antibodies in patient samples have been used in veterinary diagnostics for a little over a decade. These quantitative assays offer several advantages compared to classical serological assays, like a lower limit of detection, less background, and a broader linear quantification range, all of which improve test accuracy. The simultaneous multiplex analysis of a patient's serological response to several specific antigens also improves the diagnostic result interpretation. This influences treatment and management decisions and often allows for a quantitative follow-up as treatment response evaluation. In this review article, we discuss examples of 3 diagnostic multiplex assays for antibody detection in veterinary patients: the Lyme Multiplex assay, the Canine Brucella Multiplex assay, and the Equine Herpesvirus Type-1 Risk Evaluation assay. In addition, multiplex assays for immune response markers, like soluble cytokines, chemokines, or other inflammatory proteins, have recently become available. Currently, these assays are mainly used as clinical research tools to broadly evaluate immune activation and/or inflammation during a variety of infectious and noninfectious diseases. Quantitative cytokine and inflammatory marker multiplex assays have the potential to identify sensitive immune biomarkers for maintaining health and performance in veterinary animals.

Evaluating the performance of VP1 expressed in baculovirus and Escherichia coli expressed from Senecavirus A in pig using an ELISA.

Evaluating the performance of VP1 expressed in baculovirus and Escherichia coli expressed from Senecavirus A in pig using an ELISA.

The efficacy and safety of rituximab in adult patients with steroid-dependent or frequent relapsing nephrotic syndrome: A retrospective study.

The efficacy and safety of rituximab in adult patients with steroid-dependent or frequent relapsing nephrotic syndrome: A retrospective study.

Detection of human IgG antibodies against Mycoplasma genitalium using a recombinant MG075 antigen.

Mycoplasma genitalium is the second most common sexually transmitted bacterial infection after chlamydia. The long-term consequences of the infection are still under investigation, but reliable tools for monitoring exposure by detection of antibodies have been lacking specificity due to the presence of cross-reacting antibodies to the closely related Mycoplasma pneumoniae. Here, we describe a novel diagnostic antigen with promising sensitivity (87%) and specificity (95%) based on testing of sera from patients with PCR-confirmed M. genitalium infection and children with and without M. pneumoniae infection, respectively. The MG075F1 antigen was expressed in Escherichia coli, and the recombinant antigen was used in a western line-blot. Due to the insolubility of the antigen, harsh denaturing conditions were needed, making an enzyme-linked immunosorbent assay (ELISA) format impossible. Future work should explore shorter fragments or protein engineering to allow for assay designs better suited for high-throughput screening.

Development and validation of a blocking ELISA for measurement of rabies virus neutralizing antibody.

Enzyme-linked immunosorbent assay (ELISA) is an effective approach for monitoring herd immunity of animals against rabies but not recommended to detect neutralizing antibody response of individual animals in the framework of international travel since its insufficient agreement with the globally recognized rabies virus (RABV) neutralizing tests. In this study, a blocking ELISA to specifically detect anti-RABV neutralizing antibody was developed using the purified RABV glycoprotein (G) expressed in HEK293T cells as coating antigen, and a labeled anti-G neutralizing monoclonal antibody as the blocking antibody. The overall agreement between the blocking ELISA and fluorescent antibody virus neutralization test in detection of clinical serum samples (dogs = 658; cats = 508) was 97.43% (1,136/1,166), with a diagnostic specificity of 95.63% (219/229) and a diagnostic sensitivity of 97.87% (917/937). Further comparison with the commercial ELISA kits and inter-laboratory validation showed that the blocking ELISA had excellent specificity, sensitivity, and reproducibility. In conclusion, the developed method is a potential tool as an alternative to the virus neutralization test for the detection of canine and feline rabies neutralization antibodies following vaccination.IMPORTANCEThis study establishes a blocking enzyme-linked immunosorbent assay (ELISA) for detecting rabies neutralizing antibodies in dogs and cats, demonstrating high sensitivity, specificity, and no cross-reactivity. This method provides a reliable alternative to conventional neutralization assays, facilitating efficient large-scale rabies vaccination assessment, and thereby strengthening global rabies control efforts.

Production and purification of recombinant proteins VP2 and VP3 of the <i>Alongshan</i> virus of the <i>Jingmenvirus</i> group and evaluation of their immunochemical properties

Introduction. Alongshan virus is a representative of the unclassified group of Jingmenviruses (Flaviviridae), which is detected in Ixodes persulcatus, Ixodes ricinus ticks and various mosquito species in Russia, China, Finland and France. Unlike traditional orthoflaviviruses, the Alongshan virus genome is represented by 4 positive-sense RNA segments. The first and third segments of the genome encode proteins homologous to proteins of the replicative machinery of orthoflaviviruses, the remaining segments encode putative structural proteins that have no known homologues: segment 2 — VP1a (envelope protein), VP1b and NuORF; segment 4 — VP2 (capsid) and VP3 (membrane). Human cases of Alongshan virus-associated disease have been described. The aim of this study is to develop a system for expression and purification of recombinant VP2 and hydrophilic site VP3 proteins to test their antigenic properties. Materials and methods. Miass527 strain of Alongshan virus was used to produce hyperimmune mouse sera and recombinant proteins in a bacterial expression system. Bioinformatic analysis of sequences encoding target proteins and genetically engineered cloning were carried out in this study. Western blotting and enzyme immunoassay (ELISA) were performed to control the results. Results. Recombinant proteins of Alongshan virus have been used in a laboratory diagnostic test system to determine the presence of antibodies to the virus. The obtained recombinant VP2 protein is able to detect antibodies in all tested sera of infected mice, as well as antibodies in human sera both in Western blotting and in enzyme immunoassay. At the same time, antibodies to the recombinant region of VP3 protein were detected irregularly in antiviral immune sera. Conclusion. The detection of antibodies to Alongshan virus in patients confirms the necessity for further investigation of this group of viruses.

Method development for the detection of anti-drug antibodies against a therapeutic peptide: assay format selection.

Monitoring immune responses to therapeutic peptides with endogenous counterparts is crucial for evaluating drug safety and efficacy. In this paper, we focused on the selection of an optimal assay format to develop a sensitive, robust, and drug-tolerant immunoassay for the detection of anti-drug antibody (ADA) against a therapeutic peptide. We assessed distinct ADA assay formats for preclinical and clinical studies, such as direct binding with labeled protein A/G, direct binding with labeled multiple species-specific antibodies for detection, bridging and affinity capture elution (ACE) formats. The assay formats were evaluated based on multiple assay parameters including sensitivity, drug tolerance, individual matrix variability and inter-assay precision. Overall, direct binding assay with labeled protein A/G for detection, which utilized less labeled peptide drug and achieved desired sensitivity and drug tolerance, is appropriate for preclinical studies. Bridging assay is more suitable format to support clinical studies as bridging assay has less assay variability than ACE assay. This study highlighted advantages and limitations of each ADA assay format for peptide drugs and evaluated the performance of different assay formats in the assay development process to aid in the selection of the best fit-for-purpose assay formats for preclinical and clinical phases.

Detection of antinuclear antibodies: a survey done by the European Organsation for External Quality Assurance Providers in Laboratory Medicine.

A questionnaire was sent to immunology laboratories worldwide by the European Organisation for External Quality Assurance Providers in Laboratory Medicine to evaluate current practice with regard to how antinuclear antibodies (ANAs) are routinely tested in clinical laboratories. In total, 494 questionnaires were returned from 44 countries. Of these, 379 provided sufficient information to be included in the analysis. Indirect immunofluorescence on HEp-2 cells is still the most common method to test ANAs and is used by 330 of our 379 respondents. The most common (60%) screening dilution is 1:80, followed by 1:160 (15%) and, in equal amounts, 1:40 and 1:100 (8% each). In most laboratories, ANA-positive samples are further diluted to an end titer of 1:1280 (40%), 1:2560 (21%), 1:5120 (16%), or 1:640 (19%). An increasing number of laboratories (178/330) use the International Consensus on ANA Patterns (ICAP) nomenclature to describe the immunofluorescence pattern on HEp-2 cells. In countries with the most respondents, the percentage of laboratories accredited to EN International Organization for Standardization (ISO) 15189 (in Britain, BS EN ISO 15189, which is a British standard as well as a European standard as well as an ISO standard with identical content) is between 8% (Belgium) and 60% (France). There was no difference in the portion of accredited laboratories between university hospitals, nonuniversity hospitals, and private laboratories. Indirect immunofluorescence continues to be the most frequently used technique for ANA testing in laboratories. The increasing number of laboratories using the ICAP classification reflects an ongoing harmonization of describing ANA patterns on HEp-2 cell substrates.

Engineering a Proximity Biosensor via Constitutional Dynamic Chemistry

AbstractAffinity binding‐induced DNA assembly is a fundamental principle for designing proximity biosensors for sensitive and wash‐free protein detection and imaging. However, current design strategies for these biosensors face an intrinsic trade‐off between binding affinity and background signal. Here, we demonstrate that this intrinsic issue can be addressed by using constitutional dynamic chemistry (CDC) as a guiding principle in the rational design of proximity biosensors. As exists in a dynamic equilibrium, the constitutional dynamic network (CDN)‐based proximity biosensors can be adjusted to maximize the affinity to the target protein while minimizing non‐specific interactions that contribute to background signals. By further detecting the ratio of agonist to antagonist within the CDN, we also significantly improved assay robustness, enabling the sensitive detection of antibodies in complex matrices such as human serum. With the high affinity, low background, and high robustness, we anticipate that our CDN‐based design strategy will find wide applications in biosensor development. Our study also opens the possibility to engineer protein‐responsive synthetic systems with complex dynamic behaviors and functions.

Development of a double-antigen sandwich ELISA for rapid and accurate detection of antibodies against Capripoxvirus.

In this study, a double-antigen sandwich ELISA was developed based on the 122 protein with high immunogenicity and conservation during early viral replication, aiming to detect antibodies against Capripoxvirus. This method features high sensitivity and specificity, is cost-effective, simple, reproducible, and suitable for extensive testing. It can detect antibodies at an early phase and serves as a powerful tool for epidemic monitoring, prevention, and control.

Engineering a Proximity Biosensor via Constitutional Dynamic Chemistry.

Affinity binding-induced DNA assembly is a fundamental principle for designing proximity biosensors for sensitive and wash-free protein detection and imaging. However, current design strategies for these biosensors face an intrinsic trade-off between binding affinity and background signal. Here, we demonstrate that this intrinsic issue can be addressed by using constitutional dynamic chemistry (CDC) as a guiding principle in the rational design of proximity biosensors. As exists in a dynamic equilibrium, the constitutional dynamic network (CDN)-based proximity biosensors can be adjusted to maximize the affinity to the target protein while minimizing non-specific interactions that contribute to background signals. By further detecting the ratio of agonist to antagonist within the CDN, we also significantly improved assay robustness, enabling the sensitive detection of antibodies in complex matrices such as human serum. With the high affinity, low background, and high robustness, we anticipate that our CDN-based design strategy will find wide applications in biosensor development. Our study also opens the possibility to engineer protein-responsive synthetic systems with complex dynamic behaviors and functions.

Longstanding, undiagnosed, highly replicative hepatitis B virus reactivation in the presence of high levels of anti-HBs antibodies

Abstract Introduction Kidney transplant recipients are among the populations at risk for Hepatitis B Virus (HBV) reactivation, and close monitoring is needed for its early detection. Methods We describe a case of HBV reactivation in a patient who underwent kidney transplantation more than 30 years ago, with a known serological profile of past HBV infection. Results Reactivation occurred as a highly replicative infection that went undiagnosed for 7 years due to negative results for HB surface antigen (HBsAg) and high levels of anti-HBs antibodies. Viral genome sequencing showed a high number of mutations in the major hydrophilic region of HBsAg that could explain such a profile. Discussion This case highlights the usefulness of frequent and systematic HBV viral load testing in patients at risk of reactivation, with anti–hepatitis B core antibodies, regardless of HBsAg detection, aminotransferases, and anti-HBs antibody levels.

Clinical and diagnostic significance of isolated detection of IgA antibodies to deamidated gliadin peptides in IgA nephropathy patients

Background. Immunoglobulin A nephropathy (IgAN) is a serious medical problem reported to be one of the most common causes of terminal renal failure. Current research increasingly focuses on the role of intestinal mucosa-associated lymphoid tissue (MALT) in IgAN pathogenesis, especially under the effect of food antigens, such as gluten. Patients with IgAN often have IgA antibodies to deamidated gliadin peptides (antiDGP IgA). Studying their isolated carriage can help in the development of new diagnostic and therapeutic methods aimed at regulating intestinal immune response and managing the highly active course and progression of IgAN.Objective: To establish the clinical and diagnostic role of anti-DGP IgA in IgAN patients for the development of additional personalized clinical approaches and optimization of treatment strategies.Material and methods. A total of 105 IgAN patients aged 18 to 64 years participated in the controlled, prospective, comparative, cohort study. Demographic, anamnestic, clinical, and treatment data were used. The blood of patients was tested for celiac-specific antibodies: anti-DGP IgA, IgA antibodies to tissue transglutaminase (anti-TTG IgA), IgA antibodies to endomysium. As a result, patients were divided into two groups depending on the presence of anti-DGP IgA: the main group (n=20) comprising IgAN patients with detected antibodies and the control group (n=85) consisting of patients seronegative for celiac antibodies. One patient was seropositive for both anti-DGP and anti-TTG IgA.Results. As compared to the control group, the patients of the main group exhibited higher IgAN activity, which was assessed in terms of morning proteinuria (0.96 [0.70–1.60] g/l; p=0.005), daily proteinuria (1.50 [0.70–2.50] g/day; p=0.014), erythrocyturia (20.00 [15.00–25.00] per high power field; p=0.015), as well as levels of systolic (147.65±12.06 mm Hg; p=0.001) and diastolic (94.35±12.78 mm Hg; p=0.006) blood pressure. The detection of anti-DGP IgA was associated with a high concentration of serum IgA (4.35±1.06 g/l; p < 0.001). The direct correlation between anti-DPG IgA and IgA (ρ=0.247; p=0.020) can most likely be attributed to the hyperreactivity of IgA-producing B-lymphocytes of the intestinal mucosa in response to gluten. In the main group, the risk of a 50% decrease in the estimated glomerular filtration rate or progression to terminal renal failure within 5 years after the performed renal biopsy was statistically significantly higher than in the control group (15.05% [9.32–20.91] vs. 7.99% [4.97–11.73]; p=0.015).Conclusion. The obtained results indicate the possibility of using anti-DGP IgA as a potential risk marker for IgAN progression. Further studies into the effect of food antigens on the immune response in IgAN opens up new prospects for the development of effective treatment methods.

Development and laboratory validation of an electrochemiluminescence ELISA technique for measuring infliximab concentrations and anti-drug antibodies.

Development and laboratory validation of an electrochemiluminescence ELISA technique for measuring infliximab concentrations and anti-drug antibodies.

Design of hybrid aptamer-molecularly imprinted polymer nanoparticles for selective binding of oxidized low-density lipoprotein in an ELISA-mimic system.

Design of hybrid aptamer-molecularly imprinted polymer nanoparticles for selective binding of oxidized low-density lipoprotein in an ELISA-mimic system.