Amprion, Inc.

Carly Farris

Luis Concha

Bret Holguin

Yihua Ma

Benedikt K Vollrath

Russell M Lebovitz

Frank Espin

Hieu Huu Nguyen

Inert matrices for use with α-synuclein seed amplification assays (αS-SAAs) are provided. The inert matrices accurately reflect the absence of misfolded αS protein when used as a negative control, in the form of no, perceptively low, or delayed αS substrate self-aggregation, yet will readily permit aggregation of the αS substrate with seeds when used as a positive control. The inert matrices may be used to screen for αS-SAA reagent competence. The inert matrices may be used to dilute samples taken from peripheral biological matrices. Finally, the inert matrices may be used as a diluent for serial dilutions of αS-SAA samples, to enable semi-quantitative versions of αS-SAAs.

Inert matrices for qualitative and semi-quantitative seed amplification assays

An expression vector is provided for production of human alpha-synuclein (αS) protein or a conservative variant thereof that exhibits a decreased tendency to self-aggregate in an αS seed amplification assay (SAA). The expression vector comprises a nucleic acid sequence coding for human αS protein or a conservative variant, the nucleic acid sequence comprising codons that have been optimized to produce human αS protein or a conservative variant when expressed by a host cell such as. The codons have been optimized to avoid amino acid misincorporation in the expressed protein. Methods for purification of the expressed protein are also provided.

Alpha-synuclein substrates and methods for making and using the same

A method is provided for determining the presence of soluble, misfolded α-synuclein protein in a biological sample. The method comprises contacting the biological sample with a pre-incubation mixture, the pre-incubation mixture comprising: a monomeric α-synuclein protein; a buffer composition; a salt; and an indicator, to form an incubation mixture. An incubation cycle is conducted on the incubation mixture in the presence of either a silicon nitride bead or a borosilicate glass bead having a diameter of from about 1 mm to about 5 mm. The method further comprises determining if a detectable amount of misfolded α-synuclein aggregate is present in the biological sample.

Alpha-synuclein detection using beads

An expression vector is provided for production of human alpha-synuclein (&#x3b1;S) protein or a conservative variant thereof that exhibits a decreased tendency to self-aggregate in an &#x3b1;S seed amplification assay (SAA). The expression vector comprises a nucleic acid sequence coding for human &#x3b1;S protein or a conservative variant, the nucleic acid sequence comprising codons that have been optimized to produce human &#x3b1;S protein or a conservative variant when expressed by a host cell such as. The codons have been optimized to avoid amino acid misincorporation in the expressed protein. Methods for purification of the expressed protein are also provided.

A method is provided for determining the presence of soluble, misfolded &#x3b1;-synuclein protein in a biological sample. The method comprises contacting the biological sample with a pre-incubation mixture, the pre-incubation mixture comprising: a monomeric &#x3b1;-synuclein protein; a buffer composition; a salt; and an indicator, to form an incubation mixture. An incubation cycle is conducted on the incubation mixture in the presence of either a silicon nitride bead or a borosilicate glass bead having a diameter of from about 1 mm to about 5 mm. The method further comprises determining if a detectable amount of misfolded &#x3b1;-synuclein aggregate is present in the biological sample.

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San Diego, CA, United States of America

Carly Farris