| goal | | Tentative target based on TREX-KO heart 0.25 pmol/mL | criteria (LLoQ<10pg/mL (=10 nM)) (IN 202004 monthly update) |
| Modality | | LC/MS (should be main effort | MSD (Meso Scale Discovery, a type of immunoassay), |
| 202002 | | | Regarding MSD-based assay development, standard curve was determined as approx. 43 pg/mL of LLoQ |
| 202004 | | | High sensitive MSD assay system (LLOQ= 13.4 pg/mL) has been developed, mostly achieved to the criteria (LLoQ<10pg/mL) - will further improve sensitivity |
| 202006 | | | high sensitive MSD assay system (LLoQ= 7.2 pg/mL) has been developed. Next step is assay verification using human pooled CSF samples and TREX1KO mouse brain |
| 202007 | | (Nishi Toshiya 20200729: Please make it clear that we are still in the process of signal identification both in ELISA and MS, strictly speaking. I would still be a bit cautious unless further confirmation.
I believe the putative cGAMP peak was also detected in TREK-WT mice. Mentioning only TREK-KO can be misleading.
The 10ng/mL CSF data is still something tentative. (?, because we didn't characterize species?)
One more thing from my end is that I have finally embarked on a feasibility study for cGAMP aptabody screening this month.
Bioanalysis method by LC/MS/MS is under development to quantify the concentration in human CSF and TREX1KO mouse brain. | In the preliminary experiment of immunoassay using MSD system, the concentration of cGAMP in human (commercial, normal) CSF was about 10 ng/mL (tentative). (js: so NSTM measures human CSF before mouse brain) To make it clear that MSD count truly reflects 2'3'-cGAMP, it will be done to confirm the specificity of 2'3'-cGAMP for this immunoassay protocol. |
| 202008 | | Method development still ongoing (mice brain -> human CSF) (js: so DMPK measures mouse brain before human CSF) | We performed the below experiments in August. The data are under analysis. I expect to share these updates within the next week.
1. Compare MSD counts among isoform (2'2'-, 2'3'-, 3'3'-cGAMP)
2. 1st trial of mice brain in MSD |
| 202009 MU | ↑ Cgas protein in Park2 KO Ipsc-Dan & midbrain of paraquat mice, but not in brains of Parki KO (45 w) mice | (still) working on LC/MS method for cGAMP. Method optimization (e.g. extraction method, mobile phase, surrogate matrix exploration) is on-going to achieve better sensitivity for mouse brain samples. | We are also working on MSD assay for cGAMP in human CSF. In this month, we confirmed that the antibody, which we have used in the current MSD method, is very specific for 2'3'-cGAMP.
To confirm the specificity of antibody for 2'3'-cGAMP, standards of 2'2'-, 2'3'-, 3'3'-cGAMP were determine in the current MSD method. In the result, LLOQ of 2'3'-cGAMP was about about 100 times lower than that of 3'3'-cGAMP, and LOD of 2'3'-cGAMP was about 60 times lower than that of 3'3'-cGAMP. LLOQ and LOD of 2'2'-cGAMP were not calculable, suggesting that antibody used in the current method has a high affinity for 2'3'-cGAMP. |
| My summary | | cGAMP: Method optimization for LC/MS assay is ongoing to achieve better sensitivity in mouse brain samples. | With MSD assay, the specificity of antibody to 2'3'-cGAMP was confirmed compared to 2'2'- and 3'3'-cGAMP, with a plan to determine cGAMP concentration in CSF derived from healthy volunteers. |
| 20201007 외 | | | created a prototype assay method for cGAMP in human CSF.
Confirmed CSF-cGAMP level in HV (mean±SD: 4871.3±790 pg/mL, N=5)
30uL of CSF aliquot. the cGAMP concentration in human CSF was 7.22 nM (n=5, mean), that is slightly higher than we expected.
this method can be used for various studies like patient stratification, in vitro and in vivo pre-clinical assay for cGASi PJ.
* For In vitro: we have not assessed cGAMP in iPSC. We have succeeded to quantify only human CSF cGAMP. If needed, we can transfer our protocol to DDU. I think they need to make some modification for in vitro assay.
* Mice: DMPK is working on LCMS (for brain, not CSF). If they want to quantify mice CSF cGAMP as well as mice brain, we can support them (i.e. transfer the protocol, check the detectability of cGAMP in mice CSF).
* Where did get HV CSF?: -We purchased individual CSF from PrecisionMed
*: 1nM=674 pg/mL
LLoQ of 2'3'-cGAMP was 0.24 nM (Quantitative range: 263.3-269600 pg/mL)
- Next steps:
- Modification of protocol to be robustness
- Evaluate cGAMP in HV and patients (indication is under discussion): js: we need sPD (for stratification purpose), Parkin-PD (To confirm pathway activation), PINK1-PD (To confirm pathway activation),
- Kamiguchi: this is BFA. |
| 202010 MU | | LC/MS-based assay for CSF and brain homogenate is under development as well | Using MSD assay, CSF-cGAMP in HV (individual) was determined as 4871.3±790 pg/mL (=7.22 nM). That was consistent with pooled CSF-cGAMP level (5192.8 pg/mL). |
| 202011 | | assay development for cGAMP using LC/MS is on-going for CSF and brain samples. | Regarding MSD assay, we performed the preliminary experiment to confirm the feasibility of our assay system. But we do not have any updates that we should report in the monthly one page.
Will do PD CSF (individual) |
| 202101 | | Development of LC/MS method for cGAMP measurement in CSF and tissue samples is on-going. However, we have some update just recently and now are summarizing it to share with the team | MSD assay system for CSF-cGAMP has been established and measured in healthy volunteer (4871.3±790 pg/mL, N=5). We will move on to measure CSF-cGAMP in PD patients. |
| 20210217 m meeting | | ELISA csf. 진짜 CGAMP PEAK 인지 불명 (peak separation 잘 안됨), Plan: optimization 더 할거다, nanoLC(더 잘 separation)할거다, MICE (TREX1 KO) brain 볼거다. | We will move on to measure CSF-cGAMP in PD patients. as feasibility study (20 CSF samples will be delivered at the end of March). |
| 202102 | | DMPK team is also working hard-to establish cGAMP detection method by LC-MS/MS. They will use the nano LC to have the better separation of cGAMP | The TSHO MBM team ~~also successfully established cGAMP detection method by MSD even though there might be room to improve the assay condition. They are~~ is planning to measure cGAMP in PD patient's CSF as a pilot feasibility study to test the MSA cGAMP assay developed in house (20 CSF samples will be delivered at the end of March). |
| 202103 | | Development of LC/MS method for cGAMP measurement in CSF and tissue samples is on-going. | MSD assay system for CSF-cGAMP has been established and measured in healthy volunteer (4871.3±790 pg/mL, N=5). We will move on to measure CSF-cGAMP in PD patients. |