Development and validation of a sensitive sandwich ELISA against human PINK1
Bibliographic record
Abstract
The ubiquitin kinase and ligase PINK1 and PRKN together label damaged mitochondria for their elimination in lysosomes by selective autophagy (mitophagy). This cytoprotective quality control pathway is genetically linked to familial Parkinson disease but is also altered during aging and in other neurodegenerative disorders. However, the molecular mechanisms of these mitophagy changes remain uncertain. In healthy mitochondria, PINK1 protein is continuously imported, cleaved, and degraded, but swiftly accumulates on damaged mitochondria, where it triggers the activation of the mitophagy pathway by phosphorylating its substrates ubiquitin and PRKN. Levels of PINK1 protein can therefore be used as a proxy for mitochondrial damage and mitophagy initiation. However, validated methodologies to sensitively detect and quantify PINK1 protein are currently not available. Here, we describe the development and thorough validation of a novel immunoassay to measure human PINK1 on the Meso Scale Discovery platform. The final assay showed excellent linearity, parallelism, and sensitivity. Even in the absence of mitochondrial stress (i.e. at basal conditions), when PINK1 protein is usually not detectable by immunoblotting, significant differences were obtained when comparing samples from patient fibroblasts or differentiated neurons with and without PINK1 expression. Of note, PINK1 protein levels were found increased in human postmortem brain with normal aging, but not in brains with Alzheimer disease, suggesting that indeed different molecular mechanisms are at play. In summary, we have developed a novel sensitive PINK1 immunoassay that will complement other efforts to decipher the roles and biomarker potential of the PINK1-PRKN mitophagy pathway in the physiological and pathological context. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECL: electrochemiluminescence; ELISA: enzyme-linked immunosorbent assay; iPSC: induced pluripotent stem cell; KO: knockout; LLOQ: lower limit of quantification; MSD: Meso Scale Discovery; PD: Parkinson disease; p-S65-Ub: serine-65 phosphorylated ubiquitin; Ub: ubiquitin; ULOQ: upper limit of quantification; WT: wild-type.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.004 | 0.003 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.002 | 0.002 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".