Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I
Bibliographic record
Abstract
BACKGROUND: With myocardial infarction (MI), cardiac troponin is released from the heart into circulation, where it can be detected with immunoassays independently quantifying cardiac troponin I (cTnI) or cTnT. There is, however, no single immunoassay that sequentially probes the posttranslational modification status of cTnI or directly characterizes whether circulating cTnI is bound to cTnC and/or cTnT. Here we describe the development of a qualitative immunoassay to directly probe the primary and ternary structure of circulating cTnI through diffractive optics technology (dotLab System, Axela). METHODS: Anti-cTnI antibody 8I-7 was immobilized on a patterned sensor to capture cTnI. One or more detector antibodies were sequentially introduced to probe for amino acid sequence integrity or phosphorylation status of cTnI, or its association with cTnC and/or cTnT. Respective immunocaptures were recorded as real-time diffractive intensities (DIs), and the DI differences were analyzed. Each immunodetection was independent of the others but was done in a single sequential assay. RESULTS: This diffraction-based immunoassay successfully characterized cTnI. The unamplified assay determined whether cTnI was degraded at N-terminus and/or C-terminus or phosphorylated. Sequential application of multiple detector antibodies without an antibody-stripping step enables real-time interrogation of 5 different epitopes of cTnI, or direct detection of the cTn complex (cTnI-cTnC-cTnT) in a single sequential assay. Finally, this assay was optimized with amplification to directly detect circulating cTnI bound to cTnC and cTnT in serum from an MI patient. CONCLUSIONS: The dot Immunoassay is the first qualitative sequential immunoassay to address the direct interactions of the troponin subunits and various modified forms of cTnI.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.003 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 teacher head, 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".