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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F67985904%3A_____%2F18%3A00498970" target="_blank" >RIV/67985904:_____/18:00498970 - isvavai.cz</a>

Výsledek na webu

—

DOI - Digital Object Identifier

—

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.nWe aimed to develop and verify Luminex xMAP assay for mHTT detection based on commercially available antibodies.nWe 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.n

Název v anglickém jazyce

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

Popis výsledku anglicky

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.nWe aimed to develop and verify Luminex xMAP assay for mHTT detection based on commercially available antibodies.nWe 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.n

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

30403 - 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])

Projekt

<a href="/cs/project/LO1609" target="_blank" >LO1609: Modely závažných lidských onemocnění: Traumatické poškození míchy, Huntingtonova choroba, melanom a neplodnost</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů