- CASP1 cleaved
- Human CSF CASP1 cleaved : s or r plex accelerate?) or accelerate Quanterix?
- Rat CSF and plasma assay available but should Katy develop cleaved-specific assay?
- GSDMD: current Katy’s rat assay is not suitable (and not specific to cleaved so we should develop new Rat CSF and plasma (Katy)?, tell Janaky to add rat to Proteina (CSF and …) both?)
[Sortell]
| Marker | SN | Striatum | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| AAV-aSyn | PFF | AAV-aSyn | PFF | |||||||
| AAV-1mo | AAV-2mo | PFF 1mo | PFF 2mo | PFF 6mo | AAV-1mo | AAV-2mo | PFF 1mo | PFF 2mo | PFF 6mo | |
| NLRP3 | ✓1.2x | |||||||||
| ASC | ||||||||||
| Cleaved caspase-1 | = | ✓7x (p10,p20 both) | nd | = | ✓1.9x | |||||
| Casp1 FL | Nd | |||||||||
| GSDMD cl | nd | = | nd | ✓1.8x (31kDa) | ✓1.5x | ✓1.2x | ||||
| GSDMD FL | ↓ | ↑ (~1.3 이것인듯) | nd | |||||||
| TSPO | = | = | = | = | ||||||
| IL-1b | = | nd | ✓1.6x | ↓ (~40%) | = | = | ||||
| TH (decrease) | ✓23% | ✓38% | ✓68% | ✓30% | ✓12% | ✓25% | ✓30% | |||
| CSF casp1 | TBD TBD | TBD | ↑ (~1.3) | = | = | ND | ||||
| CSF IL-18 | TBD | TBD | = | =( ↓ ) | =( ↓ ) | |||||
| Plasma Casp1 | = | = | ND | |||||||
| Plasma IL-18 | = | = | ||||||||
| Rat | Mouse | Human | NHP | |
|---|---|---|---|---|
| MSD S-plex (custom) | MSD V-plex | MSD S-plex | MSD V-plex | |
| LOD | ~0.17 pg/ml | ~0.11 pg/ml | 0.02 pg/ml | 0.05 pg/ml |
| LLOQ | ~0.6 pg/ml | ~0.72 pg/ml | 0.098 pg/ml | 0.65 pg/ml |
| Endogenous Plasma range | 0-2.3 (avg. 0.8pg/ml) | 0.46-2.4 pg/ml (avg 0.86) | 0.026-0.6 pg/ml (avg 0.16) | 0.08-2.17 (avg 0.4) (Rhesus) 0.12-20.2 (avg 0.33) (cyno) |
| Endogenous serum range | NI | 1.13-3.95 pg/ml (avg 2.3) | 0.032-1.1 pg/ml (avg 0.082) | 0.12-32.3 (avg 1) (Rhesus) 0.09-5.39 (avg 0.22) (cyno) |
| Endogenous CSF levels | BLOD (TSD) | BLOD (TSD) | 0-2.5 pg/ml (avg 0.06) | NI |
| MRD | 2 | 2 | undiluted | 2 |
| Volume required | 20ul | 25ul | 25ul | 25ul |
| Rat, 20220630 | Plasma | CSF |
|---|---|---|
| IL-18 | 12ul | 20ul |
| Caspase-1 | 20ul | 5ul |
| mouse | Rat-SD | Rat - Fisher | ||
|---|---|---|---|---|
| IL-1b | brain | Normal whole brain homogenate was also BLOD | I will order a new rat IL-18 kit (approx.. 2-3 weeks lead time), and measure both IL-1b and IL-18 in Pauls 6-month Fischer rats: plasma and CSF, and potentially brain (whole, or dissected if there is a particular region that is of interest.) | |
| IL-1b - CSF | I am able to measure CSF IL-1b in pooled normal CSF (approx. 2 mo) at just above the LOQ ~ 10pg/ml | I will order a new rat IL-18 kit ... | ||
| IL-1b - Plasma | Plasma for normal and 6-OHDA (age?) was BLOD. BLOD in MSD assay, Quanterix SP-X barely measurable baseline | I will order a new rat IL-18 kit ... | ||
| [MSD S-PLEX] 이제부터 develop 해야 하는 것임 for $75k. But they already have an antibody pair that works for the less sensitive assay, so antibody screening wouldn't be needed: R-PLEX assay uses two mouse monoclonal Abs, and the V-PLEX assay uses a mouse mAb and a goat polyclonal. | ||||
| IL-18 | brain | is easily measurable in normal plasma, CSF, and whole brain homogenate. | I will order a new rat IL-18 kit ... | |
| IL-18 csf | is easily measurable in normal plasma, CSF, and whole brain homogenate. | I will order a new rat IL-18 kit ... | ||
| IL-18 Plasma | is easily measurable in normal plasma, CSF, and whole brain homogenate. | I will order a new rat IL-18 kit ... | ||
Workflow
Run kit with provided standards in buffer:
Added QCs, Dilution series of pooled plasma, CSF, and brain homogenate; estimate LLOQ and MRD (→ if there is reasonable dilutional linearity)
| if an assay requires ul volume | 100 | a mouse can provide ul volume (considering pipetting dead volume) | 8 |
|---|---|---|---|
| dilution | then the assay requires ul volume | we need x animals for the one data point | |
| 2 | 50 | ||
| 4 | 25 | 3.125 | |
| 8 | 12.5 | 1.5625 | |
| 16 | 6.25 | 0.78125 |
spike recovery if possible (many times the provided calibrator is not concentrated enough to do, depending on endogenous levels)
Because rodent CSF often has invisible blood contamination, if plasma levels are higher than CSF levels, → I will do whole blood spiking experiments with CSF to evaluate potential for plasma protein contamination
If assay is reproducible and fairly robust, I will measure individual animals, usually within the context of a pharmacology study where normal/untreated animals are included.
Longitudinal
| Univ of Alabama | ||||||
|---|---|---|---|---|---|---|
|
• introduction: 개인, pd general (aSyn, lysosome, mitochondria, inflammation!), we are preclinical, so clinical trials is not our scope Transfer of full subject-level, item-level data on all available subjects for clinical scale assessments including imaging, fluid biomarkers • Summary statistics (mean, median, range, standardized response means in the target population) for the following: • 우리가 돈내서 cohort recuit 를 촉진 (for future data) • Consultancy agreement? • Il-1b: <30%, • DPA-71: simplified reference tissue model (SRTM) was used to estimate the binding potential (BP), with the cerebellum cortex serving as the reference region • we observed an increase in the NLR in PD subjects • Send sample? • Other company? • concept of peripheral immune cell infiltration early in PD71. 판단: CDA 필요? Consultancy agreement 필요? | ||||||
| Udall cohort | Baseline: Participants were enrolled between November 2018 and December 2021 | 30 patients are currently 3 years after enrollment | F/U? ANNualy | Enrolment | finalized. Currently only recruiting controls that dropped out (10; 1 PD patient died. | |
| DPA-714 | PD 30, HC 37 | 120 PD patients (? HC 합쳐서 아닌가?) of which 40 HAB were scanned | No, because of cost | |||
| CSF | Pd 25, HC 19 | No, because invasive | ||||
| plasma | PD 62, HC 56 | Likely (how many?) | ||||
| PBMCs (high quality) | ||||||
| MDS-UPDRS | likely | |||||
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Sortell column 8 (Striatum AAV-1mo) | nd (Cleaved caspase-1) | The cell appears to read “nd” but a small numeric annotation may be partly cropped above. |
| GSDMD FL Striatum row | trailing cells empty | Several lower cells in this row are blank in the source; could not confirm whether this represents missing data or ”=“. |
| Univ of Alabama bullet | Il-1b: <30%, | The percent value follows “Il-1b” with a < 30%-like glyph; could read ≤30%. |
| Udall row “Enrolment” | 1 PD patient died. | Numeric prefix could read either 1 or 1.; statement boundary uncertain. |
| LOD/LLOQ table NHP cells | 0.08-2.17 (avg 0.4) (Rhesus) 0.12-20.2 (avg 0.33) (cyno) | The two species-specific ranges share a single cell with a small space separator; comma may be intended. |