Robert Neely

Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer’s Disease

Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.

An integrated multiplatform bioanalytical strategy for antibody–drug conjugates: a novel case study

BACKGROUND The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.

Evolution of fit-for-purpose biomarker validations: an LBA perspective

Biomarker ligand-binding assays need to be validated for use on clinical studies in the drug development process. There is not one single guidance to cover all types of biomarker assays and their intended uses. Therefore, it is up to the scientist to piece together a validation strategy based on published papers and other sources. Shown here is a summary of what to take into consideration during a validation and how to apply it for use in the drug development process.

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.

Reproducibility of a fully automated, chemiluminescent, beta-amyloid 42 assay

upon a detailed unified SOP for the INNO-BIA AlzBio3 (RUO; Innogenetics nv, Belgium) multiplexing xMAP assay. Using the same assay batch, a set of ten CSF pool samples and two buffer based control samples were analyzed using ready-to-use calibrators. A fresh aliquot of each sample was tested in quadruplicate in three independent assay runs (only 2 runs for center C). The goal of the study was to determine the with-in and interlaboratory variability of the assay for hTau, Ab 1-42, and P-Tau 181P after implementation of the unified SOP. Results: The CSF analyte concentrations showed a strong correlation between all centers (R> 0.95; regression center versus overall center mean). For the CSF samples, the total inter-center variability over the 8 runs performed (%CV) ranged between 12.4 22.9% (hTau), 8.1 13.1% (Ab 1-42), and 6.8 13.2% (P-Tau 181P). Across the 10 CSF samples, the mean within-laboratory variation ranged between 1.8% and 11% for all analytes. The variability observed for the control samples was even lower (max. 4.5%).Conclusions: Implementation of a unified SOP in the three laboratories resulted in an acceptable inter-laboratory variation of analyte concentrations for CSF and control samples. Careful documentation of the critical parameters of the test procedure and rigorous adherence to a detailed instruction of use clearly leads to highly comparable CSF analyte concentrations between experienced centers.

Stability performance of a fully automated, chemiluminescent, beta-amyloid 42 assay

P1-137 STABILITY PERFORMANCE OFA FULLY AUTOMATED, CHEMILUMINESCENT, BETA-AMYLOID 42 ASSAY Karen Ackles, George Green, Holly Soares, Flora Berisha, Robert Neely, Irina Baburina, Salvatore Salamone, Daniel Kozo, Shari Jackson, Anne Tweedie, Deb Byrne, Lisa DiMagno, Ortho Clinical Diagnostics, Rochester, New York, United States; Bristol Myers Squibb, Princeton, New Jersey, United States; Bristol Myers Squibb, Wallingford, New Jersey, United States; Bristol Myers Squibb, Lawrenceville, New Jersey, United States; Bristol-Myers Squibb, Princeton, New Jersey, United States; Saladax, Bethlehem, Pennsylvania, United States; Saladax Biomedical, Inc., Bethlehem, Pennsylvania, United States; Saladax Biomedical, Bethlehem, Pennsylvania, United States. Contact e-mail: kackles@its.jnj.com

Fit for purpose Clinical validation of a multiplex immunoassay for the measurement of Tau, p-Tau and Abeta 42

Methods: For this purpose, organotypic hippocampal cultures (OHCs) were exposed to Aß1-42 (2 mM) for 48 hours with or without curcumin (0.5, 1, 5 and 10 mM). We also analysed the effect of LY294002 (5 mM), an inhibitor of phosphoinositide-3-kinase (PI3-K) pathway in OHCs exposed to Aß1-42 (2 mM) and curcumin (10 mM). Cell death was measured by propidium iodide uptake in the slices and some cell signaling pathways were investigated by performing Western blot assay with specifics antibodies. Moreover, we measured the synaptophysin expression, involved in the regulation of synaptic plasticity, by Western blot assay. Results:Our results show that Aß1-42 peptide caused about 30% of cell damage in hippocampal slices, a significant increase when compared to controls cultures. The treatment with 5 and 10 mM of curcumin decreased the cell death significantly. The curcumin treatment prevented the decreased in synaptophysin expression after exposure to Aß peptide. Aß1-42 neurotoxicity resulted in an increased in phosphorylated (Ser45) ß-catenin and a decreased in ß-catenin immunocontent and the curcumin treatment prevented this ß-catenin destabilization. Additionally, the curcumin (10 mM) neuroprotection was prevented by LY294002. Immunoblotting revealed that curcumin induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of glycogen synthase kinase-3ß (GSK-3ß). Conclusions: These results reinforce the neuroprotective effect of curcumin and add some evidence that its mechanism may involve the PI3-K pathway and ß-catenin signaling, a key transducer of the Wnt signaling pathway. We also provide interesting perspectives on the use of our model in the study of this disease process.