Absolute quantitation of serum antibody reactivity using the Richards growth model for antigen microspot titration

In spite of its pivotal role in the characterization of humoral immunity, there is no accepted method for the absolute quantitation of antigen specific serum antibodies. We devised a novel method to quantify polyclonal antibody reactivity, which exploits protein microspot assays and employs a novel analytical approach. Microarrays with a density series of disease specific antigen were treated with different serum dilutions and developed for IgG and IgA binding. By fitting binding data of both dilution series to a product of two generalized logistic functions, we obtained estimates of antibody reactivity of two immunoglobulin classes simultaneously. These estimates are the antigen concentrations required for reaching the inflection point of thermodynamic activity coefficient of antibodies and the limiting activity coefficient of antigen. By providing universal chemical units, this method may improve standardization of serological testing, the quality control of antibodies and the quantitative mapping of antibody-antigen interaction space.

HIV Antibody Profiles in HIV Controllers and Persons With Treatment-Induced Viral Suppression

IntroductionLow HIV viral load is associated with delayed disease progression and reduced HIV transmission. HIV controllers suppress viral load to low levels in the absence of antiretroviral treatment (ART). We used an antibody profiling system, VirScan, to compare antibody reactivity and specificity in HIV controllers, non-controllers with treatment-induced viral suppression, and viremic non-controllers.MethodsThe VirScan library contains 3,384 phage-displayed peptides spanning the HIV proteome. Antibody reactivity to these peptides was measured in plasma from a Discovery Cohort that included 13 elite controllers, 27 viremic controllers, 12 viremic non-controllers, and 21 non-controllers who were virally suppressed on ART. Antibody reactivity to selected peptides was also assessed in an independent cohort of 29 elite controllers and 37 non-controllers who were virally suppressed on ART (Validation Cohort) and in a longitudinal cohort of non-controllers.ResultsIn the Discovery Cohort, 62 peptides were preferentially targeted in HIV controllers compared to non-controllers who were virally suppressed on ART. These specificities were not significantly different when comparing controllers versus viremic non-controllers. Aggregate reactivity to these peptides was also high in elite controllers from the independent Validation Cohort. The 62 peptides formed seven clusters of homologous epitopes in env, gag, integrase, and vpu. Reactivity to one of these clusters located in gag p17 was inversely correlated with viral load set point in an independent cohort of non-controllers.ConclusionsAntibody reactivity was low in non-controllers suppressed on ART, but remained high in viremic controllers despite viral suppression. Antibodies in controllers and viremic non-controllers were directed against epitopes in diverse HIV proteins; higher reactivity against p17 peptides was associated with lower viral load set point. Further studies are needed to determine if these antibodies play a role in regulation of HIV viral load.

Development of a Rapid Diagnostic Kit for Congestive Heart Failure Using Recombinant NT-proBNP Antigen

Background and Objectives: In patients with congestive heart failure, brain natriuretic peptide (BNP) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) are released due to excessive heart muscle expansion; they can be used for the early detection, progress monitoring, and treatment of congestive heart failure. Recently, considerable efforts have been made to develop an NT-proBNP-based biomarker for detecting heart failure. This study attempts to develop a rapid and accurate congestive heart failure diagnostic kit using NT-proBNP. Materials and Methods: A new gene based on NT-proBNP was selected, recombined, and expressed in Escherichia coli strains, and then monoclonal antibodies were produced using the hybridoma technique. Additionally, antigen-antibody reactivity was confirmed using indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the first pair and full-strip pair tests were conducted to select candidate clones; these were applied to a rapid diagnosis kit based on gold conjugates and compared with other currently available antigens. Results: NT-proBNP-based antigens with high specificity and monoclonal antibodies were produced, and the optimal antigen-antibody reactivity was confirmed using indirect ELISA. The first pair and full-strip pair tests were performed to select the optimal candidate clones, and a rapid diagnosis kit with excellent reactivity was developed by applying these to a rapid diagnosis kit based on gold conjugates. Conclusions: The development of this rapid diagnosis kit with excellent performance in congestive heart failure is expected to improve disease management by providing an early assessment of the risk of heart failure.

SARS-CoV-2 anti-nucleocapsid assay performance in healthcare workers at baseline and 6 months

Introduction Serological SARS-CoV-2 assays have an important role in guiding the pandemic response. This research aimed to compare the performance of 2 antinucleocapsid assays. Methods Serum from 49 HCWs was analysed at baseline and 6 months using the Abbott diagnostics SARS-CoV-2 IgG assay and the Roche Diagnostics Elecsys Anti-SARS-CoV-2 total antibody assay. Results At baseline, 14/49 participants (29%) demonstrated antibody reactivity using the Abbott assay. At 6 months, 4/14 participants (29%) continued to demonstrate reactivity. A total of 14/49 (29%) participants had detectable antibodies at baseline using the Roche assay. In total, 13/14 (93%) of participants demonstrated antibody reactivity at 6 months. The Abbott assay showed a statistically significant difference in the signal-to-threshold values of baseline reactive samples when repeated at 6 months (p = 0.001). This was not seen with the Roche assay (p = 0.51). Conclusion In this small study, the Roche Diagnostics Elecsys Anti-SARS-CoV-2 total antibody assay appears superior in performance to the Abbott diagnostics SARS-CoV-2 IgG assay in accurately detecting participants with a history of confirmed COVID-19 disease at 6 months follow-up. This finding should be born in mind in the planning of future seroprevalence studies, especially when considering the use of anti-nucleocapsid assays.

Diverse Humoral Immune Responses in Younger and Older Adult COVID-19 Patients

As numerous viral variants continue to emerge in the coronavirus disease 2019 (COVID-19) pandemic, determining antibody reactivity to SARS-CoV-2 epitopes becomes essential in discerning changes in the immune response to infection over time. This study enabled us to identify specific areas of antigenicity within the SARS-CoV-2 proteome, allowing us to detect correlations of epitopes with clinical metadata and immunological signals to gain holistic insight into SARS-CoV-2 infection.

Mass Spectrometry-Based Proteomic and Immunoproteomic Analyses of the Candida albicans Hyphal Secretome Reveal Diagnostic Biomarker Candidates for Invasive Candidiasis

Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.

Mass Spectrometry-based Proteomic and Immunoproteomic Analyses of the Candida albicans Hyphal Secretome Reveal Diagnostic Biomarker Candidates for Invasive Candidiasis

Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.