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The HSPCs engrafted and soon differentiated into macrophages in key regions of the mice's brain and spinal cord where they appeared to transfer wildtype FXN into deficient neurons and muscle cells.
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.