Quantification continued, end-point dilution + Calibration curve, application, Utility (Sidoroy 2023 Syn-target / RRK2-targeted / Aa-targeted), MJF workshop 20220421, Sources of aSyn (Cell/Brain/Recombinant/Synthetic + Aβ Figure 1)

Quantification (continued)

	end-point dilution assay for SD50	comparison of lag phases or times-to-threshold
Calibration curve		known nSyn vs LAG
application	(original sample (we 존재하는 seed 의 estimate 시간 (so only disease samples are used)	Buffer Q 일감

Utility

(Sidoroy, 2023)

	SAA positive ratio	utility
Syn target Ts	91.3% in iPD	• To exclude SAA-negative patients • To exclude pathology in PrP, Tau, TDP-43, Amyloid beta
RRK2 targeted Ts	67.5% in LRRK2-PD	• To exclude SAA-negative patients
Aa-targeted Ts	95.5% in GBA-PD	• To exclude SAA-negative patients Rationale: Since GBA Parkinson’s disease is overwhelmingly α-syn SAA positive, thus, a negative result would raise questions about a Parkinson’s disease diagnosis • Tx verify whether GBA PD is really spreading prone
prodromal PD	To check the paper	Tx verify whether SAA assay is phasing
somal PD		Tx verify whether lysosomal PD is really spreading prone

MJF workshop 20220421

Can WE USE AV~ IN our msa trial?

• RT-Quic quantitative 되면 people with more aSyn → faster seeding time

Utility: stratification BM reflect tax LB aSyn pathology, so differentiate from PD vs HC, PD vs MSA, PRODROMAL 에 쓰일 수 있다. Progression 예는 마사용 Form↑ LB aSyn antibody 에도 잘 수 있을 듯.

• Prodromal pid 들 plasma bm 사용해서 더 잘 identify 할 수 없나 (eg dat scan loss ~ 20%)
• Clinical outcome 과 correlation 짚는 bm 만 필요하다고 알리지. Updrs 가져가 heterogeneous & variable 하므로 long term prognosis/outcome (eg cognition fall 등 track 위는 bm 이 필요하니까)

SZ: It can identify early prodromal patients and differentiate from MSA because of the shape of the curve (lower maximal signal or plateau). But I doubt it will be quantitative because all the data I have seen with dilutions of the same sample result in the same curve. Amprion have done this for MSA and PD using MJFF samples.

Sources of aSyn

	Cell-derived AB	Brain-derived AB	Recombinant AB	Synthetic
(category)	CHO cells / viable transfection with human APP/PS1 bearing the V717F mutation / cells	Human brain (Transgenic mouse brain (eg KP-1 KO)) → tissue homogenization → centrifugation → ultracentrifugation; (or stem mass affinity chromatography); HPLC	E. coli	Cell-grown AβP human
Method	made using recombinant DNA techniques, E. coli; homogenization → sucrose gradient fractionation (→ i)IF ii) filter retardation assay using antibodies against α-syn, p-aSyn, ubiquitin or p62 so this is called 'LB-...'			synthesis

Figure 1 caption: Common sources of beta-amyloid peptide (Aβ) used for modelling Alzheimer’s disease (AD)-related processes in vitro and in vivo.