20240722_183206

Analyte	Date	Content
Gasdermin D		Some paper used ELISA kit from not-trustworthy supplier (from my experiences). I think the detection of cleaved GSDMD by antibody for full-length is insufficient. The condition between western blotting and immunoassay is very different. (e.g. denatured vs conformational, one antibody vs 2 non-competing antibody.). Abcam kit ordered → initial result horrible PUKBB and PrecisionMed data coming in Dec. NBB data coming in Dec.
aSyn fragment	20231016	[Ab] Large fragment (1-121) Small fragment (122-140) C-terminal (NEAYEMPSEEGYQDYEPEA) nitrosylated C-terminal (NEAYEMPSEEGYQDYEPEA) phosphorylated C-terminal (NEAYEMPSEEGYQDYEPEA) ab3309 MP used. E4U2F XP® Rabbit mAb #51510 잠재, 신호: carrier-free version of product #51510 → for conjugation to magnetic beads available without BSA and Azide (CS#45083), which may be used for conjugation to magnetic beads, Biotin conjugated preferred? Detects FL too. Detects FL too. (Joe Palandra: digestion 힘기니까 이건 걱정 x) If necessary, one option would be to ask CS to produce antibody in PBS or appropriate buffer (대안: Francis to check the price of ~1 mg production of the antibody to which options would be better. cellsignal #51510 cellsignal #45083 BSA-and-azide-free [aSyn] • Walter has purified FL and large fragment (1-121), but not C-term (121-140, due to small size and difficulty). On the other peptide is available. → Walter to send C-term fragment peptide (1~ 2 mg) to Joe, and full-length and large fragment of α-syn just in ... From E.coli is OK. [plan] • antibody generation campaign is not necessary. • Joe Palandra will test the peptides in matrix samples. He has HV CSF in hand. feasibility assay, and if everything goes well, 3~4 weeks, and pilot date will be around Dec.
	20231207	C-terminal fragment (NEAYEMPSEEGYQDYEPEA) nitrosylated C-terminal fragment (NEAYEMPSEEGYQDYEPEA) phosphorylated C-terminal fragment (NEAYEMPSEEGYQDYEPEA) peptides Assay sensitivity (buffer 겠지) sensitivity lower limits of 100 pg/ml(충분?) do seem achievable for all peptides, efforts to reduce further will likely require either additional sample volume or more sensitive LC-MS platforms such as nanoflow. Detection? Quantification? - buffer with the peptides 100 ngml O Standard curve to show signal (intensiity) is proportional to the peptide concentration? O O Antibody Cell signaling antibody does bind two of the 3 C-terminal peptides (C-term and nitrosylated C-term) X (→there are commercial antibodies that were developed against phosphorylated position that work even with this smaller sequence) however optimal binding was observed using a combination of cell signaling and TAK-341 antibodies. Early indications is that extraction recovery appears to be greater than 70% using this antibody pair. phosphorylated position that work even with this smaller sequence Sample extraction (immunoprecipitation, in multiple matrices including artificial CSF (electrolytes, but no protein), 0.5% BSA/PBS, diluted human CSF Successful X (because the antibody can't bind?) artificial CSF + spike (from 100 pg/ml) spike successfully detected X Human CSF + spike Verbal: (from 100 pg/ml) spike successfully detected, 적어도 there is no matrix effect! Human CSF : w/o spike le endogenous Endogenous peptides not detected. → 아마 Nanoflow 등으로 sensitivity 높인후 test 할 것 (PD and HC, BioIVT) Additional assessments may include nanoflow, methionine oxidation, and additional CSF matrices depending on available resourcing
	20240110	Working on nanoflow: should improve sensitivity down to 10-20 pg/ml (note: otake: total csf aSyn is ~100 pg/ml) Slide 2: antibody enrichment x, Slide 3: CST Ab + TAK-341 combined. Plan) Feb get PD CSF → test (required volume 200 uL) Apr: improve CST Antibody to recognize phosphorylated aSyn peptide [20240126] we will need to pool them to increase sample volume, which will also increase sensitivity, reduce sample amounts and allow them to be processed in a single batch run
	20240220	. I was able to analyze these samples last week and unfortunately I was not able to detect any measurable amounts of the alpha syn C-terminal fragment in any of the samples including the AAV A-syn + veh where we would have expected the values to be the greatest. I was able to decrease the lower limit of quantitation to 7.5 pg/mL but unfortunately that did not prove to be low enough. I also did want to mention that I had obtained an isotope labeled reference internal standard prior and did use it in the sample run when I spiked a fixed concentration into every sample. By doing so it allowed me to track whether the antibody pulldown worked and the consistency throughout the entire run. The results confirmed that I was able to successfully measure this in all the rat CSF samples thus there is added confidence to support the findings from the run. Human: , I don't have any human data besides a human CSF pool in which I also did not find any detectable levels.
	20240306	αSyn fragment detection in mouse CSF samples Assay lower limit of quantitation is 7.5 pg/mL. Rat CSF samples did not show any measurable levels of α-synuclein C-terminal peptide though internal standard was successfully extracted αSyn fragment detection in human CSF samples (HC): Pooled sample CSFs were analyzed, but no peak was observed Is there any possibility that rat aSyn may inhibit human aSyn detection/enrichment? => Labeled human aSyn which was added to all samples prior to antibody enrichment could be recovered, so it is unlikely. Plan: positive samples (Atuka mouse striatum homogenate, SH-SY5Y pellet transfected with eGFP-labelled which over-expresses different parts of aSyn, CSF from future TSPO PET STUDY in AAV aSyn rat) with p-aSyn (MP will send MJF's antibody list) measurement attempt ) → PD CSF samples (~200 uL required)
	PNMB	20231207	[AAV aSyn Rat] Brain: SN not available, but PR to look into striatum, PR to source antibody (ELISA, eg testing) CSF, Plasma: Katy : evaluate kits → test [human] We cannot use somascan, so to qualify katy's MSD ECL assay in PD vs HC CSF,

Quanterix:

NLRP3 pathway SIMOA assay by MJFF-Quaterix

PHASE 1: ANTIBODY PAIR FINDING, SPECIFICITY TESTING, AND INITIAL ASSAY OPTIMIZATION:

plan:

• Identify Ab pairs for NLRP3, GSDMD, ASC, CASP1, IFNb, and HMGB1.
• Sensitivity testing using recombinant protein and specificity testing using KO cells.
• Spike recovery and dilution linearity testing in healthy control human CSF and plasma samples.

timeline:

• Start date = October 2021
• Estimated end date = June 2022

PHASE 2: ASSAY VALIDATION FOR PRECLINICAL AND EARLY CLINICAL EVALUATION:

plan:

• Development of multiplex panel for NLRP3 analytes listed above.
• Assay sensitivity, dilution linearity, stability, (intra/inter-) robustness/reproducibility testing, etc.
• Assay testing in patient samples (eg NLRP3 gain-of-function mutation carriers, PD patient samples)

timeline:

• Anticipated Start date = August 2022
• Estimated end date = January 2023

PHASE 3: GLP ASSAY DEVELOPMENT FOR LATE-STAGE CLINICAL TRIAL USE

plan:

• Transfer of the assay to a separate CRO for GLP-certification and reevaluation

timeline:

• TBD

Progress

Date	Content		Validation (for preclinical & early clinical)	Development for late-stage	Commercial
20220419	other collaborators are welcome, and additional samples sets are welcome. During our meeting, I brought up that other samples existed and thought that we could potentially use samples like yours to pre-qualify the assay. we could potentially include Takeda in future discussions, but at a minimum, keep you updated on the progress and eventual availability of the assays for sample testing
	optimization
	NLPR3	Cleaved Caspase-1	ASC, N-terminal GSDMD		I-1B
20220212	Plan: optimization (eg. linearity) → CSF Screening → → three options: i) Quanterix in house ii) share protocol with industry iii) commercial kit LLQQ, what is MJF need? Freeze-thaw, volume required?

Uncertain Spans

location	transcription	uncertainty
aSyn fragment / 20231016 antibody sub-table / [aSyn] bullet	reads Walter has purified FL and large fragment (1-121), but not C-term (121-140, due to small size and difficulty). On the other peptide is available. → Walter to send C-term fragment peptide (1~ 2 mg) to Joe, and full-length and large fragment of α-syn just in ... From E.coli is OK.; the trailing just in ... token is partly clipped at the column boundary.	low confidence on the trailing token.
aSyn fragment / 20231016 antibody sub-table / digestion 힘기니까 이건 걱정 x	the Korean phrase reads as digestion 힘기니까 이건 걱정 x and likely is digestion 힘드니까 이건 걱정 x (handwritten variant); preserved verbatim.	low confidence on 힘기 vs 힘드.
aSyn fragment / 20240220 entry / α-syn token	reads the alpha syn C-terminal fragment and the AAV A-syn + veh; the inconsistent α-syn / A-syn / aSyn tokens are preserved verbatim.	source typography preserved; not corrected.
Progress sub-table / column placement	the rows optimization, NLPR3 / Cleaved Caspase-1 / ASC, N-terminal GSDMD and the Plan: optimization → CSF Screening → three options: rows form a sub-table whose column-by-analyte placement is partly clipped; the I-1B token in the rightmost cell is preserved verbatim though it likely denotes IL-1β.	low confidence on column placement and I-1B vs IL-1β.