Protocol Code: SCF-BAP-PCR-N6K7-EPI-GMS-0001
Classification: GLP-Compatible Multi-Omics Assay Suite for Translational Deployment
I. OVERVIEW
This document defines CRO/NICU-ready assay protocols for quantifying:
- Immunomic markers (ELISA)
- Metabolomic / redox markers (LC-MS/MS)
- Transcriptomic markers (RNA-seq panels)
Aligned to SCF-PCR BRAID™ indices (PTI, ISI, RI).
II. ELISA PANEL — IMMUNOMIC & PROTEOMIC ASSAYS
Target Panel
- IL-1β, TNF-α (Preventative)
- IL-6, HMGB1 (Restorative)
- BDNF (Plasticity marker)
2.1 SAMPLE COLLECTION
Parameter | Condition |
Matrix | Plasma / CSF |
Volume | 50–200 µL |
Anticoagulant | EDTA |
Processing | Centrifuge 1500 g, 10 min, 4°C |
Storage | −80°C |
2.2 REAGENTS
- Pre-coated ELISA plates (capture antibody-specific)
- Detection antibody (biotinylated)
- Streptavidin-HRP
- TMB substrate
- Stop solution (1N H₂SO₄)
2.3 ASSAY PROCEDURE
Step 1 — Plate Preparation
- Add 100 µL standards + samples
- Incubate 2 hrs at RT (or overnight at 4°C)
Step 2 — Washing
- Wash 3–5× with PBS-T (0.05% Tween-20)
Step 3 — Detection Antibody
- Add 100 µL detection antibody
- Incubate 1 hr at RT
Step 4 — HRP Conjugation
- Add 100 µL streptavidin-HRP
- Incubate 30 min at RT
Step 5 — Signal Development
- Add TMB substrate (100 µL)
- Incubate 10–15 min (dark)
- Add stop solution
Step 6 — Readout
- Measure absorbance at 450 nm
2.4 DATA OUTPUT
Parameter | Output |
Sensitivity | pg/mL |
CV | <10% |
Dynamic range | 10–1000 pg/mL |
SCF LINKAGE
- IL-1β / TNF-α → PTI (Preventative trigger)
- IL-6 / HMGB1 → RI (Recovery index)
III. LC-MS/MS PANEL — METABOLOMIC & REDOX ASSAYS
Target Panel
- Lactate
- ATP / ADP ratio
- NADH / NAD⁺ ratio
- Glutamate / GABA
- CoQ10
3.1 SAMPLE PREPARATION
Parameter | Condition |
Matrix | Plasma / CSF |
Volume | 50–100 µL |
Extraction | Methanol precipitation (3:1 MeOH:sample) |
Centrifugation | 15,000 g, 10 min |
Supernatant | Collect for analysis |
3.2 LC CONDITIONS
Parameter | Setting |
Column | C18 reverse-phase |
Flow rate | 0.3 mL/min |
Gradient | Water (0.1% FA) / ACN |
Run time | 10–20 min |
3.3 MS CONDITIONS
Parameter | Setting |
Mode | ESI (+/−) |
Analyzer | Triple quadrupole |
Detection | MRM (multiple reaction monitoring) |
3.4 KEY TRANSITIONS (EXAMPLES)
Analyte | m/z Transition |
Lactate | 89 → 43 |
ATP | 506 → 159 |
NADH | 664 → 408 |
Glutamate | 148 → 84 |
GABA | 104 → 87 |
3.5 DATA OUTPUT
Parameter | Output |
Quantitation | µM / nM |
Sensitivity | Low nM |
CV | <15% |
SCF LINKAGE
Marker | Index |
Lactate ↑ | PTI / RI |
ATP ↓ | RI |
NADH/NAD⁺ imbalance | PTI |
Glutamate ↑ | ISI |
IV. RNA-seq PANEL — TRANSCRIPTOMIC PROFILING
Target Genes
- SCN8A (Nav1.6)
- KCNQ2 / KCNQ3 (Kv7)
- CACNA1H (T-type Ca²⁺)
- GABRA1 (GABA receptor)
- BDNF
4.1 SAMPLE COLLECTION
Parameter | Condition |
Source | Whole blood (PAXgene) / CSF cells |
RNA stabilization | Immediate |
Storage | −80°C |
4.2 RNA EXTRACTION
- Kit: silica column-based or magnetic bead
- Yield: ≥100 ng total RNA
- Quality: RIN ≥7
4.3 LIBRARY PREPARATION
Step | Method |
mRNA enrichment | Poly-A selection |
Fragmentation | Enzymatic |
cDNA synthesis | Reverse transcription |
Adapter ligation | Indexed adapters |
4.4 SEQUENCING
Parameter | Setting |
Platform | Illumina |
Read length | 75–150 bp |
Depth | 20–50 million reads/sample |
4.5 DATA ANALYSIS PIPELINE
- Alignment (STAR / HISAT2)
- Quantification (FPKM / TPM)
- Differential expression analysis (DESeq2)
4.6 OUTPUT METRICS
Gene | Interpretation |
SCN8A ↑ | Hyperexcitability |
KCNQ2 ↓ | Reduced stabilization |
CACNA1H ↑ | Burst propensity |
BDNF ↑ | Plasticity activation |
SCF LINKAGE
Gene | Index |
SCN8A ↑ | PTI / ISI |
KCNQ2 ↓ | PTI |
CACNA1H ↑ | ISI |
BDNF ↑ | RI |
V. INTEGRATED MULTI-OMIC ASSAY WORKFLOW
Parallel Processing Pipeline
- Blood / CSF collection
- Split into:
- ELISA (cytokines)
- LC-MS (metabolites)
- RNA-seq (gene expression)
Time-to-Result
Assay | Time |
ELISA | 4–6 hrs |
LC-MS | 6–12 hrs |
RNA-seq | 24–48 hrs |
VI. QUALITY CONTROL
Controls
Type | Example |
ELISA | Standard curve (8-point) |
LC-MS | Internal standards (isotopic) |
RNA-seq | Spike-in controls |
Acceptance Criteria
- ELISA CV <10%
- LC-MS CV <15%
- RNA integrity RIN ≥7
VII. CLINICAL INTEGRATION (PCR BRAID LINK)
Biomarker → Action
Assay Result | Action |
IL-1β ↑ | ↑ Preventative dosing |
Glutamate ↑ | Activate Curative layer |
ATP ↓ | Intensify Restorative layer |
VIII. SCF STRATEGIC ADVANTAGE
- Multi-omic integration → real-time decision making
- Direct linkage to dosing algorithms (PCR Braid)
- Enables closed-loop therapeutic systems
IX. INTEGRATED SCF LOGIC SUMMARY
High-resolution biomarker quantification + multi-omic integration + PCR-linked decision logic → precision-guided seizure management
MASTER REGISTRY INDEX
- SCF-BAP-PCR-N6K7-EPI-GMS-0001 — Biomarker Assay Protocols
- SCF-MOBP-PCR-N6K7-EPI-GMS-0001 — Biomarker Panel
- SCF-PCRB-N6K7-EPI-GMS-0001 — PCR Braid Strategy
- SCF-DA-PCR-N6K7-EPI-GMS-0001 — Dosing Algorithms
- SCF-DDI-N6K7-EPI-GMS-0001 — DDI Modeling
- SCF-SEF-MD-0001 — Synergistic Evaluation Framework
If you want next, I can generate:
- FDA companion diagnostic submission package
- CLIA lab implementation protocol
- Multiplex assay design (single-run panel)
- Point-of-care biomarker device concept (NICU integration)