Background: Early and accurate detection and staging is critical to managing Alzheimer’s disease (AD) and supporting clinical trials. Cerebrospinal fluid (CSF) biomarkers for amyloid-β peptides, tau species, and various neurodegenerative and inflammatory analytes are leading the way in this regard, yet there is room for improved sensitivity and specificity. In particular tau is known to be present in many different fragments, conformations, and post-translationally modified forms. While the exact tau species that might best reflect AD pathology is unknown, a growing body of evidence suggests that forms with high levels of phosphorylation in the mid-region may be especially enriched in AD. Objective: Develop an assay for measuring p217tau in CSF. Methods: Here we describe the development and validation of a novel sELISA for measuring CSF tau species containing phosphorylation at threonines 212 & 217, aka p217 + tau, using the PT3 antibody. Results: While the analyte is present at extremely low levels the assay is sufficiently sensitive and specific to quantitate p217 + tau with excellent precision, accuracy, and dilution linearity, allowing good differentiation between diagnostic subgroups. The p217 + tau measurements appear to track AD pathology better than the commonly used p181tau epitope, suggesting superior diagnostic and staging performance. Finally, the assay can also be configured to differentiate antibody-bound versus antibody-free tau, and therefore can be used to measure target engagement by p217 + tau-targeting immunotherapeutics. Conclusion: The assay for measuring p217 + tau described here is highly sensitive, accurate, precise, dilution linear, and shows good potential for identifying and staging AD.

Background: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer’s disease. Objective: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. Methods: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. Results and Conclusion: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer’s disease.

A group of six mouse monoclonal antibodies (MoAbs) with the unusual lambda-type light chain were generated by fusion of NS-1 cells with splenic cells derived from BALB/c mice immunized with crude extracts of Bermuda grass pollen (BGP). Four of them were IgG1, one was IgG2b, and one was IgG3. Binding inhibition assay showed that they recognized the same (or very similar) epitope. Using sera from BGP-allergic patients, it was found that the specific binding between the IgE antibodies and the MoAb 26-11-fixed antigen could be blocked by MoAb 26-11 itself and another MoAb 9-13 in a dose-dependent manner. It appears that the epitope recognized by the lambda-type MoAbs is a human IgE-binding antigenic determinant. Further physico-chemical analyses showed that this epitope was stable under heat but sensitive to treatments of sodium periodate and proteinase K. Results from these studies indicate that this unique epitope which leads to the generation of lambda-type MoAbs is part of a glycoprotein.

Developing variant vaccines or multivalent vaccines is a feasible way to address the epidemic as the SARS-CoV-2 variants of concern (VOCs) posed an increased risk to global public health. The spike protein of the SARS-CoV-2 virus was usually used as the main antigen in many types of vaccines to produce neutralizing antibodies against the virus. However, the spike (S) proteins of different variants were only differentiated by a few amino acids, making it difficult to obtain specific antibodies that can distinguish different VOCs, thereby challenging the accurate distinction and quantification of the variants using immunological methods such as ELISA. Here, we established a method based on LC–MS to quantify the S proteins in inactivated monovalent vaccines or trivalent vaccines (prototype, Delta, and Omicron strains). By analyzing the S protein sequences of the prototype, Delta, and Omicron strains, we identified peptides that were different and specific among the three strains and synthesized them as references. The synthetic peptides were isotopically labeled as internal targets. Quantitative analysis was performed by calculating the ratio between the reference and internal target. The verification results have shown that the method we established had good specificity, accuracy, and precision. This method can not only accurately quantify the inactivated monovalent vaccine but also could be applied to each strain in inactivated trivalent SARS-CoV-2 vaccines. Hence, the LC–MS method established in this study can be applied to the quality control of monovalent and multivalent SARS-CoV-2 variation vaccines. By enabling more accurate quantification, it will help to improve the protection of the vaccine to some extent.

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