Validation and Clinical Utility of ELISA Methods for Quantification of Amyloid-ß of Peptides in Cerebrospinal Fluid Specimens from Alzheimer's Disease Studies.

The aim of this study was to validate assays for measurement of amyloid-ß (Aß) peptides in cerebrospinal fluid (CSF)specimens according to regulatory guidance and demonstrate their utility with measurements in specimens from Alzheimer's disease (AD) studies. Methods based on INNOTEST(®)ß-AMYLOID(1-42) and prototype INNOTEST(®)ß-AMYLOID(1-40) ELISAkits were developed involving pre-analytical sample treatment with Tween-20 for reliable analyte recovery.Validation parameters were evaluated by repeated testing of CSF pools collected and stored in the same manner as clinical specimens. Intra- and interassay coefficients of variation were ≤11% and relative accuracy was within ± 10% for both analytes. Dilutional linearity was demonstrated for both analytes from a spiked CSF pool, but not from a non-spiked native CSF pool. Recovery of standard Aß peptide spikes standard ranged from 77% to 93%. No interference was observed from the investigational drugs LY2811376, LY2886721, LY3002813, or semagacestat. Aß(1-40) and Aß(1-42) were stable in CSF for up to 8 hours at room temperature and during 5 f reeze-thaw cycles from ≤−20◦C and ≤−70◦C. In frozen native CSF specimens, Aß(1-40) was mostly stable up to 3 years at ≤−70◦C, whereas stability of Aß(1-42) was limited to 221 days. Dose-dependent changes in measured CSF Aß were observed in healthy volunteers up to 36 hours after treatment with the-site cleavage enzyme inhibitor LY2886721. In conclusion, rigorous validation tests have successfully demonstrated the strengths and operational limitations of these INNOTEST(®)-based assays.They have proved to be robust and reliable tools for pharmacodynamic evaluations of investigational AD therapeutics in clinical trials.

Validation of assays for measurement of amyloid-ß peptides in cerebrospinal fluid and plasma specimens from patients with Alzheimer's disease treated with solanezumab.

The aim of this study was to validate new assays for measurement of amyloid-ß (Aß) peptides in cerebrospinal fluid (CSF) and plasma specimens in clinical studies of solanezumab according to current regulatory recommendations. Four assays based on the INNOTEST® ß-AMYLOID(1-42) and prototype INNOTEST ß-AMYLOID(1-40) kits were developed and validated. To render these assays 'solanezumab-tolerant', excess drug was added to calibrators, quality control, and test samples via a 2-fold dilution with kit diluent. Validation parameters were evaluated by repeated testing of human CSF and EDTA-plasma pools containing solanezumab. Calibration curve correlation coefficients for the four assays were ≥0.9985. Intra- and inter-assay coefficients of variation for Aß1-40 and Aß1-42 were ≤13 and ≤15%, respectively for both matrices. Dilutional linearity, within and between assays, was demonstrated for both analytes in CSF and plasma at clinically relevant dilution factors. This dilution regimen was successfully applied during Phase 3 clinical sample analysis. Aß1-40 and Aß1-42 were stable in CSF and plasma containing solanezumab at 2-8°C and room temperature for up to 8 h and during 5 additional freeze-thaw cycles from ≤-20 and ≤-70°C. Results of parallel tests on stored clinical samples using INNOTEST methods and proprietary ELISA methods were closely correlated (r2 > 0.9), although bias in reported concentrations was observed between assays. In conclusion, the modified INNOTEST assays provided (relatively) accurate and precise quantification of Aß1-40 and Aß1-42 in CSF and plasma containing solanezumab according to established consensus validation criteria. The clinical experience with these assays post validation has shown them to be robust and reliable.

Biological consequences of oxidative stress-induced DNA damage in Saccharomyces cerevisiae.

Reactive oxygen species (ROS), generated by endogenous and exogenous sources, cause significant damage to macromolecules, including DNA. To determine the cellular effects of induced, oxidative DNA damage, we established a relationship between specific oxidative DNA damage levels and biological consequences produced by acute H2O2 exposures in yeast strains defective in one or two DNA damage-handling pathways. We observed that unrepaired, spontaneous DNA damage interferes with the normal cellular response to exogenous oxidative stress. In addition, when base excision repair (BER) is compromised, there is a preference for using recombination (REC) over translesion synthesis (TLS) for handling H2O2-induced DNA damage. The global genome transcriptional response of these strains to exogenous H2O2 exposure allowed for the identification of genes responding specifically to induced, oxidative DNA damage. We also found that the presence of DNA damage alone was sufficient to cause an increase in intracellular ROS levels. These results, linking DNA damage and intracellular ROS production, may provide insight into the role of DNA damage in tumor progression and aging. To our knowledge, this is the first report establishing a relationship between H2O2-induced biological endpoints and specific oxidative DNA damage levels present in the genome.

Spontaneous DNA damage in Saccharomyces cerevisiae elicits phenotypic properties similar to cancer cells.

To determine the spectrum of effects elicited by specific levels of spontaneous DNA damage, a series of isogenic Saccharomyces cerevisiae strains defective in base excision repair (BER) and nucleotide excision repair (NER) were analyzed. In log phase of growth, when compared with wild type (WT) or NER-defective cells, BER-defective cells and BER/NER-defective cells possess elevated levels of unrepaired, spontaneous oxidative DNA damage. This system allowed establishment of a range of approximately 400 to 1400 Ntg1p-recognized DNA lesions per genome necessary to provoke profound biological changes similar in many respects to the phenotypic properties of cancer cells. The BER/NER-defective cells are genetically unstable, exhibiting mutator and hyper-recombinogenic phenotypes. They also exhibit aberrations in morphology, DNA content, and growth characteristics compared with WT, BER-defective, and NER-defective cells. The BER/NER-defective cells also possess increased levels of intracellular reactive oxygen species, activate the yeast checkpoint response pathway via Rad53p phosphorylation in stationary phase, and show profound changes in transcription patterns, a subset of which can be ascribed to responses resulting from unrepaired DNA damage. By establishing a relationship between specific levels of spontaneous DNA damage and the ensuing deleterious biological consequences, these yeast DNA excision repair-defective strains are an informative model for gauging the progressive biological consequences of spontaneous DNA damage accumulation and may have relevancy for delineating underlying mechanisms in tumorigenesis.