D07 Development of luminex xmap bead-based immunoassay for quantification of MHTT

Abstract

Background 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. Aims We aimed to develop and verify Luminex xMAP assay for mHTT detection based on commercially available antibodies. Methods 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. Motor cortex tissue was obtained from two different HD animal models, R6/2 mouse (4 weeks and 12 weeks old) and TgHD minipig (6 years old) and appropriate age-matched WT controls. We tested three different lysis buffers for brain tissue homogenate preparation: HEPES/Sucrose (no detergent), TRIS/EDTA/NP40 (non-ionic detergent only) and RIPA. 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. Results 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. Conclusion 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.