| Assay / target | Description / Reference | Result / Next step | ||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| casp1(p20) | (most likely to detect Caspase-1 p20, but monomer 인지, as a dimer 인지는 불명 이님?) | |||||||||||||||||||||||||||||||||
| Caspase-1 |
[Sebastian] "Olink Explore 384 Neurology panel will be used. 20220701 Human plasma and CSF samples from NBB will be analyzed. [absolute quantification 위해] The Focus panels are specialized custom panels built specifically for our customer needs. As they typically take anywhere from 3-6 months to develop and QC, we do have a couple of prerequisites in order to take on a project. The first is that a customer needs to have run a discovery and validation study using Olink's off-the-shelf panels to confirm their biomarker signature and provide biological ranges for their assays in the sample type/population the custom panel would be developed for. The second pre-requisite is that a customer must contractually commit to running a minimum of 1,000 samples within the first 12 months of development for that custom panel. If both of those are satisfactory, the process continues as such: • We schedule a call with Olink's Focus Team and the Customer to discuss the project and review the process and timelines • The customer signs a consent form providing access to the customer data from the off-the-shelf panels & provides a list of biomarkers & back-up biomarkers • The Focus Team conducts a feasibility analysis based upon the dilutions we run samples at as well as the signaling ranges in the customer data • We schedule a second call between the customer and Focus team to review data and provide recommendations • Once a list of biomarkers and back-up biomarkers are agreed to, we negotiate and sign a contract for the agreed to scope of work. The cost to develop and validate a panel in relative quantification is $120,000 and in absolute quantification $160,000. • The project goes into our queue and will start in the next open slot (depending on queue can be immediately, or a year+ wait). All the development and validation work is done in Sweden. • Once complete we send a validation report to the customer and then the panels are available to purchase as service or kit. Panels are typically good for a year once developed. • If the customer would like additional batches of the panel, we recommend to plan >3 months prior to expiration so Olink's team can bridge the batches together. New batch creation takes approx. 1-3 months and costs $25,000. |
(Gordon, 2018 #585) elevated circulating caspase-1 in a cohort of 21 PD patients (P = 0.023; fig. S1C) [Sebastian] "Olink panels.." (Fan, 2020 #657) PD (N=43), PBMC] WB, ↑ IL-1β & caspase-1 a | ||||||||||||||||||||||||||||||||
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Olink (LLOQ: 195.3 pg/mL) ↓ R&D systems kit (LLOQ: 6.25 pg/mL) Assay verified, but not quantifiable in (pooled) healty CSF. Antemortem: Caspase-1 in CSF was below LLOQ (i.e. <1.96 ng/mL), NBB sample 에서는 no obvious elevation |
Kit from R&D systems (LLOQ: 6.25 pg/mL) Assay verified. Results: 685.4 pg/mL in pooled healthy human plasma → i) antemortem: 20230525 에 결과 보여줬던가 불확실. ii) postmortem: 20230525 에 결과 보여줬던가 불확실. | |||||||||||||||||||||||||||||||||
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S-PLEX discussion started. (not ordered yet) Compare R-PLEX Caspase-1 assay with R&D kit. → (if it's comparable, S-PLEX development using R-PLEX antibody pair) 이건 cleaved casp1 아닌듯(?). S-PLEX 13,000K-yen, Waiting for R-PLEX kit to predict probability of success. Next step • ?위의 것들 (s or r plex)이 너무 비싸니까, 일단 activity assay 에 치중? --> 4월 striatum data 보고 결정? 20230504 Janaky: r-plex may be total casp-1, |
Next step • Deprioritize plasma measurement based on Erin'S UK biobank plasma data (but this is i) Olink (확실) NPX too, so should have high missing rate, and ii) FL). • Prioritize Caspase-1 enzymatic activity assay. | |||||||||||||||||||||||||||||||||
| Caspase-1 activity: Promega G9951 | caspase cleavage of the Z-WEHD substrate (Z-WEHD-aminoluciferin), → a substrate for luciferase (aminoluciferin) is released, → luciferase activity and generation of light by a proprietary, thermostable, recombinant luciferase. |
Promega substrate selective? YVAD-CHO Inhibitor of the luminescent signal confirms that the signal inhibition indicates that luminescence is not produced. (Ac-YVAD-CHO inhibition of the luminescent signal confirms that the signal is generated by caspase-1). Conversely, a lack of inhibition indicates that luminescence is not produced by caspase-1 activity (Figures 4 and 13). • Cell-derived caspase-1 maintains activity with a half-life >3 h in the Caspase-Glo® 1 Reagent, providing flexibility when timing luminometer readings. • Once caspase-1 is activated and released from cells, the activity is quite transient, with a relatively short half-life (18; {Walsh 2011 #2468} in buffer, Processed caspase-1 was found to be much more labile, with a half-life of 9 min. is rapidly inactivated upon formation of the mature enzyme, with a half-life of 9 min at 37 °C. (by measuring the residual enzyme activity, fig5c), rapid loss of quaternary structure, which disrupts the active site as a result of disassociation the enzyme subunits of the enzyme subunits. 20221206] Assay feasibility was confirmed in "concentrated" pooled human CSF, = 10x concentration of CSF by 10 kDa MWCO ultra-filtration (i.e. 500 uL → 50 uL) 20221206] Assay feasibility was confirmed in pooled human plasma Next step • Include assay calibrator using enzymatically active recombinant Caspase-1. • Verify the assay and pre-analytical processes (bench-top stability, dilution linearity etc) • Test individual CSF/plasma [20230125] just 'sensitivity' or 'LOD' would match better than LLOQ. I incorporated recombinant Caspase-1 (not included in the kit) to calibrate the enzymatic activity. LoD was 0.098 U/mL (=0.3pM=330fM). Then, I tested ante-mortem CSF. Detectibility was 50% (ie 7/14) in PD and 36% in HC (4/11), no room for improvement as input volume was already ~1ml. | ||||||||||||||||||||||||||||||||
| Cosomil |
202307 BDD Monthly: Fluorophore-conjugated substrate for active caspase 1 is under development at Cosomil. Takeda provided enzymatically active recombinant caspase 1 with them to accelerate their effort. Once they demonstrates improved sensitivity by 50 fold (tentative), we will start collaboration to measure caspase 1 activity in CSF from control and PD patients. (N. Narita) 20231109: Cosomil has just reported a result of the 2nd generation probe. It demonstrated assay LoD of 0.2 U/mL still requiring improvement. At the same time, we need to quickly test CSF. Negotiation with them is going to happened today PM. 20231130: We've developed 2nd generation single enzyme activity-based fluorescent probe with a vendor company (Cosomil) for caspase-1 (Ac-WEHD-GPsHMRG) with improved turnover rate that demonstrated enough detection feasibility of 0.1 U/mL. Will setup an original highly sensitive assay method with this company. 20240301: novel probe Suc-WEHD-GP-sHMRG was synthesized. It worked well in recombinant active Caspase-1 in the buffer (LoD: 0.1 U/mL), with complete inhibition by a specific inhibitor, VRT-043198. However, non-specific fluorescence was observed in human plasma and CSF that would be derived from endogenous endo-peptidase. Simultaneous use of red fluorescent probe to detect non-specific cleavage of GP sequence to discriminate Caspase-1 dependent fluorescence demonstrate its feasibility but requires further assay optimization 202403 BDD monthly) Alternative probe Suc-WEHD-Quinone methide-sHMRG was developed. However, its reaction rate was much lower than that of Suc-WEHD-GP-sHMRG and didn't generate a detectable signal from human CSF | |||||||||||||||||||||||||||||||||
| activity #2 | #2 (no substrate): Inhibitor turned probe + Biotin + Ab | Individual CSF: detectible but not quantifable, over-recovery. Plan: Fresher CSF | ||||||||||||||||||||||||||||||||
| activity #3 | Inhibitor (CM-269) |
(reverse designed) + amino-luciferin → cleaved by casp-1 → luciferase (luminescence) Inhibitor (pralnacasan = VX-740 {Poreba, 2013 #2480}) → substrate (CM-269) this is attached to luciferin → free amino-luciferin, which was subsequently oxidized by luciferase resulting in emission of light | ||||||||||||||||||||||||||||||||
| IL-1β |
-Comp Bio: OLINK [Sebastian] "Olink target cytokine", not comprehensive but targeted and measures 48 cytokines with absolute quantification ((pg/mL). Table 5. Samples tested in the S-PLEX Human IL-1β Kit (catalog screenshot, preserved as body_r04 evidence)
20220701 Human plasma and CSF samples from NBB will be analyzed. [otake 20220915] [LLOQ] Olink inflammatory 96 panel (AMP-PD): 1.5 pg/mL Olink Cytokine 48 panel (our analysis): 0.38147 pg/mL (the most sensitive) S-PLEX: 0.098 pg/mL, but detectibilty is still I will compare Olink Cytokine48 IL-1b and other sensitive IL-1b assays (S-PLEX), using commercially available AM CSF. • [20221206] world most-sensitive S-PLEX assay at LSI medience (LSIM: CRO in Japan): LLOQ: 33.2 fg/mL | |||||||||||||||||||||||||||||||||
| Olink Target48 (LLOQ: 0.38 pg/mL) |
Catalog spec [plasma] HC vs PD: 19% difference. d=0.41 → SSE: 76 per arm (1-tail, a=0.05, power 80%) Next step -Try more sensitive S-PLEX assay at LSI medience. → (20230223) CSF: majority detected but not quantifiable range, In PD csf: 6 out 21 were above 33.2 fg/mL. Mean 56.47 fg/mL, Median 25.6 fg/mL In HC csf: 2 out 11 were above 33.2 fg/mL. Mean 29.74 fg/mL, Median 25.0 fg/mL Plasma: all within quantifiable range but no difference HC vs PD [Sebastian] "Olink target cytokine", not comprehensive but targeted and measures 48 cytokines with absolute quantification (pg/mL). Olink assay is not ready for clinical trial, but helps choose appropriate off-the-shelf assay in terms of sensitivity in the future to be transferred to CRO (but it's responsibility of CBID). | |||||||||||||||||||||||||||||||||
| Olink Target48 (LLOQ: 0.95 pg/mL) |
NCNP (HC CV: 28%, PD CV 86%): and NBB data (HC CV: 44%, PD CV: 114%) available PUKBB and PrecisionMed data coming in Dec. NBB data coming in Dec. | |||||||||||||||||||||||||||||||||
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| top of casp1(p20) row | (most likely to detect Caspase-1 p20, but monomer 인지, as a dimer 인지는 불명 이님?) | Final particle “이님?” could be a typo for “이심?” / “이슴?”; the source ends with a question mark that may continue from a prior cell. |
| Cosomil row | Suc-WEHD-Quinone methide-sHMRG | ”Quinone methide” is highlighted in the source; the inserted hyphen is ambiguous. |
| activity #2 row | over-recovery. | ”over-recovery” reading is consistent with both “over recovery” and “overrecovery”; hyphen is faint. |
| IL-1β rows | ”Cell Culture Supernatant (N=4)” → AS row reads ND–AS | The end of the AS-truncated range may include a numeric upper bound that is cropped. |
| Caspase-1 activity row | at 37 °C. | The °C glyph is faint; could read at 37 C without the degree mark. |