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DEVELOPMENT OF LUMINEX XMAP BEAD-BASED IMMUNOASSAY FOR QUANTIFICATION OF MHTT
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SYSNO ASEP 0498970 Druh ASEP A - Abstrakt Zařazení RIV O - Ostatní Název DEVELOPMENT OF LUMINEX XMAP BEAD-BASED IMMUNOASSAY FOR QUANTIFICATION OF MHTT Tvůrce(i) Vodičková Kepková, Kateřina (UZFG-Y) ORCID
Vodička, Petr (UZFG-Y) ORCIDZdroj.dok. Journal of Neurology Neurosurgery and Psychiatry - ISSN 0022-3050
Roč. 89, S1 (2018), A33-A33Poč.str. 1 s. Akce Plenary Meeting of the European Huntington´s Disease Network (EHDN) Datum konání 14.09.2018 - 16.09.2018 Místo konání Vienna Země AT - Rakousko Typ akce EUR Jazyk dok. eng - angličtina Země vyd. GB - Velká Británie Klíč. slova luminex Vědní obor RIV EI - Biotechnologie a bionika Obor OECD Technologies involving identifying the functioning of DNA, proteins and enzymes and how they influence the onset of disease and maintenance of well-being (gene-based diagnostics and therapeutic interventions (pharmacogenomics, gene-based therapeutics) CEP LO1609 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy Institucionální podpora UZFG-Y - RVO:67985904 UT WOS 000446126000090 DOI 10.1136/jnnp-2018-EHDN.89 Anotace Huntington’s disease is a fatal neurodegeneration caused by an expansion of CAG repeat in the huntingtin (HTT) gene. This repeat encodes prolonged stretch of glutamines near the N-terminus of HTT protein. Precise quantification of mutant huntingtin (mHTT) protein in the brain and biofluids, such as blood or cerebrospinal fluid (CSF), is needed as a potential biomarker of HD progression and for monitoring of therapeutic trials. Luminex xMAP technology allows for highly multiplexed (up to 100 analytes) bead-based sandwich immuno-assays, which could be advantageous for simultaneous analysis of mHTT together with other analytes in precious samples.
We aimed to develop and verify Luminex xMAP assay for mHTT detection based on commercially available antibodies.
We used the combination of N-terminal anti-HTT antibody EPR5526 (Abcam) conjugated to Luminex MagPlex microspheres as a capture and MW1 (DSHB) anti-polyQ antibody conjugated to biotin as a detection antibody to detect mHTT in brain samples of model species. Different amounts of brain tissue homogenate were incubated with EPR5526 conjugated microbeads, MW1 detection antibody and R-PE conjugated streptavidine and analyzed on Luminex xMAP 200 instrument. We were able to detect mHTT signal in all R6/2 mouse and TgHD minipig samples, with linear response to total protein load (R2=0.999 for TgHD samples, R2=0.981 for R6/2 samples). Signal in WT mouse and minipig samples was close to blank (buffer only) levels. Luminex xMAP bead-based sandwich assay with antibody pair EPR5526/MW1 detects mHTT in R6/2 mouse and TgHD minipig HD models and with further optimization could serve for mHTT quantification in tissue and body fluid samples.
Pracoviště Ústav živočišné fyziologie a genetiky Kontakt Jana Zásmětová, knihovna@iapg.cas.cz, Tel.: 315 639 554 Rok sběru 2019
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