Assays of pyroptosis

analyte	method	notes	reference
NLRP3 (fragment?)		CSF could not be detected. NLRP3 in plasma was detectable but the dilution linearity did not meet the criteria. MSD: Antibody pair from abcam ELISA kit was used to develop MSD NLRP3 assay. However, as S/B ratio even at 60 ng/mL was as low as 3.3 [SMCxPro]: antibody pair (ab253423, abcam, produced by immunization with N-term fragment), full-length recombinant NLRP3 protein (ab165022, abcam, epitope unknown), 20220428: -buffer: LLOQ of 44 pg/mL (S/B ratio at 60 ng/mL was dramatically increased to 829, (250-fold higher value than that in MSD) -CSF: 0.17 ng/mL, couldn't get MRD because all diluted samples were below LLoD (ie no linearity, why?) -plasma: 4.45 ng/mL, MRD=2 - in both CSF & plasma: spike-recovery was low (~25%). Issues: low recovery (CSF & plasma), no MRD set up (CSF) where is the epitope in the full-length recombinant protein (for our standard and spike-in) and Abcam's NLRP3 ELISA's N-term fragment? Js: 다음 조건 충족이 중요할 것 -High S/B ratio in HC -Increase in PD [To Hua Zou] Sebastian (cc'd here) at TSHO needs some protein for the development of his assay: • Full length of human NLRP3 (MBP is not a problem) • Pyrin domain of human NLRP3 — I am not sure if you have this part of the protein. If we have to produce it how long it will take? • Do you have human NACHT domain too? [reason of low recovery in CSF & plasma] 1. When you spike NLRP3 protein into body fluid, it degraded. NLRP3 protein is very easy to degrade as well, especially for misfolded one. Body fluid may contain multiple protease which come from broken or dead cells. If possible, you may add some protease inhibitor cocktail in your assay system. The body fluid may contain some components (such as proteins) which associate with NLRP3 and cover the epitope. This will interfere the antibody detection of NLRP3. [Plan] - To add a protease inhibitor → (20220530) ↑ Spike recovery (~70%), but ↑ CV and so ↑ LLoQ (from 0.044 → 3 ng/mL) - Try Abcam's N-term NLRP3 protein (i) 위의 ab253423이 이것으로 produce되었으므로, ii) abcam uses this for their ELISA kit (ab274401) (instead of full-length protein), (사이즈 작으므로 misfolding-induced rapid degradatoin이 적을가능성)(epitope coverage by other unnecessary component도 적을수도 있을 수도, 아닐수도) → (20220530) CV, accuracy and LLoQ, S/B ratio, spike-recovery all improved. Next step: 1. Reproduce the data with fragmented protein 2. Try to reduce LLoQ further (include more standard curve points) (because with the current LLoQ, 2x diluted CSF was out of range) 3. Include low, medium and high spike in concentrations -Plasma 4x, 8x and -CSF neat and 2x → If the results are reproducible, we can proceed with patient samples 20220428 Seb: Used the identified antibody pair (ab253423, abcam) and full-length recombinant protein (ab165022, abcam), → standard curve → buffer-based range of our assay is 45 ng/mL - 44 pg/mL. 20220616 Seb: Still some problems with the CSF measurements. Basically the spike-recovery looks good at 2-fold diluted CSF, but not at neat CSF. But 2-fold diluted CSF is below LoQ.. We will try to concentrate the CSF, hopefully I can get those results early next week. Plasma looks promising. 20220808 Seb: Also here the signal unexpectedly decreased, but it appears to be due to a different reason, since the signal decreased in both the standard and in plasma. It seems that the cause is a much lower shelf-life of the conjugated detection antibody than we expected, with a decrease showing in already 20 days (in contrast to several months). I had secured antibody for running over 1500 samples but now I cannot use that lot. I have put a new order for antibodies, but they haven't given me an expected delivery date. [plan] i) Quanterix ii) CRO for SIMOA iii) MSD S-PLEX ($85K) 13,000K-yen, Developing SoW
		Next step • Access to Quanterix-MJFF for Simoa assay. • Develop S-PLEX assay at MSD → (20230223) SOW kicked off. → 20230306 Reagent procurement has just completed at MSD is working to confirm feasibility. → 20230530 NDU BoB#M-244 ☐ Self-antibody pair of ab274451 detected endogenous NLRP3 signal in CSF and acceptable parallelism and spike-recovery were confirmed at MSD after assay optimization. Assay procedure will be further optimized
		SMCxpro: buffer-based LLOQ of 43.9 pg/mL, but CSF below LLOQ (i.e. < 87.8 pg/mL) quantifiable in plasma (n=2) asked Janaky to ask Otake to measure (NBB) plasma samples
	s-PLEX	S-PLEX (MSD, LLOQ 48.8 pg/mL (comparable to our in-house developed SMCxPRO.) by self-antibody pair). NLRP3 in human pooled CSF was detectable but there was matrix effect and parallelism was not good. Further optimization is required. full validated assay method for human CSF will be delivered by May 15. 20231130: However, the NLRP3 level in human CSF (1) PrecisionMed, ante-morterm, n=10 HC / n=10 PD, (2) PUKBB, n=17 HC / n=30 PD, (3) NBB, n=7 HC / n=13) were either below LLOQ (24.4 pg/mL) or not different between HC and PD. Thus, NLRP3 quantification was de-prioritized and terminated.	i asked Janaky 'can we do it in parallel with CSF?'
	SIMOA (Quanterix흉내)	Pooled HC and PD CSF: BELOW lloq (possibly due to different CSF collection procedure, tubes?)
ASC		Couldn't find any publications on quantitative measurements in CSF or blood.	Expected concentration in plasma: ~1.1 µg/mL (proteinatlas) (Fan, 2020 #657)
	ELISA	[Sebastian] ELISA: Cusabio ELISA (ELISA (CSB EL019114HU)) (The capture antibody is mouse monoclonal and the detection antibody is goat polyclonal.) LLoQ: 15.6 ng/mL CRO: RayBiotech
	SMCxPro	20220412 Sebastian: I couldn't find any available antibody pair, so I will screen more antibodies. I have just ordered 5 antibodies (out of which 4 have arrived). Five different antibodies have been acquired and screening 20220701 Sebastian: MSD assay under development. Five different antibodies have been acquired and screening of a suitable antibody pair will start mid-July, using MSD-platform. 20220725 Seb: MSD assay quantitiated ASC in pooled human CSF and plasma in a preliminary experiment. 20220808 Seb: it seems that the standard is unstable and degrading even in -80°C, as we see a considerable reduction of signal in the standard curve, but not in plasma. I have ordered a new standard protein which I hope will arrive between 23rd and 30th of August. [MSD assay] 20220913 (Otake): 이미 issue resolved, Capture: PYCARD Monoclonal Antibody (OTI3E9), TrueMAB™ (OriGene - Thermo Fisher) Detection: In-house biotinylated recombinant Anti-TMS1/ASC antibody [EPR23978-28] (abcam) Secondary: SULFO-tagged streptavidin (MSD) Buffer-based LLOQ: Homebrewed MSD (LLOQ: 3.2 ng/mL (cf Buffer-based LLOQ: 0.82 ng/mL) → no difference in PrecisionMed, NBB and PUKBB samples Homebrewed MSD (LLOQ: 12.8 ng/mL) (cf Buffer-based LLOQ: 0.82 NBB data coming in Oct
+ASC speck:		-Nagar, 2021 #2087: review Plan/Strategy under development BY 마츠키 -Basiorka: plasma ASC specks by flow cytometry Detection of ASC speck requires not flowcytometer but imaging cytometer. We don't have the instrument in TSHO. But we have imaging plate reader. Do you think they can prepare THP1 lysate and share them to us? → Yes. I am sure they can prepare THP1 lysate and ship to TSHO. → Thanks. Let's keep discussion. Plan/Strategy under development BY 마츠키 (20230322) we have prepared the materials but with Proteina should be the first priority in terms of technological advance. We can evaluate the feasibility in April – May. Yasuko prepared ASC-knockout THP1 cells (https://www.invivogen.com/thp1-ko-kd-asc) and ectopic ASC expression vector to see the specificity of speck formation in flow cytometry. If we can have positive control of human fluid (e.g. CAPS), it would be great. (20231109) Trace positive signal of PE-labelled anti-ASC antibody was found in pooled control CSF and slightly increased in pooled AD CSF (just flash result and no figure sharable today). We need to optimize protocol to obtain reasonable scatter profile and show specificity. (20240119) No S/N between positive control and negative control or even blank medium in the usage of PE (phycoerythrin)-labelled anti-ASC antibody (HASC-71 clone, Biolegend, 653904) and isotype control. = high background Conclusion: It is difficult to improve S/N using flowcytometry. → Next: • Beads-based detection of insoluble ASC by flowcytometry or SMCxPRO. Note) Phycobiliproteins are fluorescent proteins derived from cyanobacteria and eukaryotic algae. Their fluorescence is much higher than chemical fluorescent probes such as fluorescein and rhodamine. R-phycoerythrin (R-PE) is one of the phycobiliproteins and has an orange fluorescence at around 578 nm, and it can be excited at 488 nm. Because of this high fluorescence, phycobiliprotein-labeled antibodies or other molecules can give greater sensitivity in flow cytometry and immunostaining
3+ASC	sPLEX, internal	Capture antibody: anti ASC (Origene, OTI3E9), Detection antibody: anti NLRP3 (Abcam, EPR23094-101) 20240119: (202401 BDD monthly) Specificity of ASC/NLRP3 complex assay was demonstrated by using ASC KO cells and their cell culture supernatant. However, • ASC/NLRP3 complex was below LoD in most CSF samples.
	SMCxPRO	202403 BDD monthly) SMCxPRO-based assay for the complex of NLRP3 and ASC demonstrated higher sensitivity than that by S-PLEX. However, its level in human CSF was still below detection limit. (Yasuko Matsuki)
se-1	Pro-caspase1		(Fan, 2020 #657)

Uncertain Spans

location	transcription	uncertainty
ASC SMCxPro Otake entry	이미 issue resolved	Korean opening 이미 is partly stylized; reading is consistent with the surrounding context.
Homebrewed MSD LLOQ	LLOQ: 12.8 ng/mL vs 3.2 ng/mL	Two separate Homebrewed MSD LLOQ values appear in adjacent paragraphs; both are recorded as visible.
analyte column at bottom	3+ASC, se-1	Leftmost analyte tags are partially cut by the row-header column boundary; reading is consistent with the visible context (NLRP3+ASC, caspase-1).