DEVELOPMENT OF LUMINEX XMAP BEAD-BASED IMMUNOASSAY FOR QUANTIFICATION OF MHTT

0498970 - ÚŽFG 2019 RIV GB eng A - Abstrakt
Vodičková Kepková, Kateřina - Vodička, Petr
DEVELOPMENT OF LUMINEX XMAP BEAD-BASED IMMUNOASSAY FOR QUANTIFICATION OF MHTT.
Journal of Neurology Neurosurgery and Psychiatry. Roč. 89, S1 (2018), A33-A33. ISSN 0022-3050. E-ISSN 1468-330X.
[Plenary Meeting of the European Huntington´s Disease Network (EHDN). 14.09.2018-16.09.2018, Vienna]
Grant CEP: GA MŠMT(CZ) LO1609
Institucionální podpora: RVO:67985904
Klíčová slova: luminex
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)

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.

Trvalý link: http://hdl.handle.net/11104/0291252