Molecular genetic analyses for detecting GAArepeat expansions within intron 1 or mutations in the FXN coding sequence itself are currently used to confirm a clinical suspicion of FRDA.
To address this limitation, Steinkellner and colleagues developed an electrochemiluminescence assay for a 96-well format to measure FXN concentrations in lymphocyte extracts (12).
We hypothesized that a duplex immunoassay for quantifying the concentrations of FXN and a control protein might be applicable to WB and dried blood spots (DBSs).
Polyclonal, anti-FXN, rabbit detector antibodies (PAC 2517), and purified recombinant human FXN isoforms ([FXN.
The amounts of FXN calibrators per well were 63 pg, 31 pg, 15 pg, 8 pg, 4 pg, and 0 pg.
81-210] represent, respectively, the least and most abundant of the 4 mitochondrial FXN isoforms in different tissues, including lymphoblasts (14).
25 ng/mL FXN and 19 [micro]g/mL CP in Assay Buffer) into microcentrifuge tubes containing 200 [micro]L Assay Buffer.
The purpose of the CP analyte was to serve as an internal control for measuring FXN, and thus a common eluent volume was selected to best match the linear response of our anti-FXN antibody pair.
The results from 4 repeat linearity experiments for recombinant FXN and CP spiked into DBSs and EDTA-containing blood eluents as measured with the duplex assay are summarized in Fig.
We observed a mean difference of 1%, with measured fingerstick FXN concentrations being generally slightly higher than for EDTA-containing blood.
We compared the response of our duplex to that of purified solutions of the recombinant FXN isoforms, [FXN.