High-Throughput Immunoassay for the Biochemical Diagnosis of Friedreich Ataxia in Dried Blood Spots and Whole Blood

Abstract

BACKGROUND Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 μL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%–13% and 9.8%–16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2–200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12–22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1–26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.

Randomized, double‐blind, placebo‐controlled study of interferon‐γ 1b in Friedreich Ataxia

“…Buccal cell frataxin levels correlate with disease severity, but they are present in very low levels, and their changes may not match neurological function . Whole blood frataxin levels largely represent levels in erythrocytes, where frataxin has a slightly different amino acid sequence and is made from a different splice variant . These levels are unlikely to correlate with neurological function.…”

Usefulness of frataxin immunoassays for the diagnosis of Friedreich ataxia

“…Protein was eluted from DBS, mixed with frataxin antibody-coupled microspheres, and analysed for frataxin content using the Luminex LX200 instrument 24. Calibration of frataxin content was performed using purified human frataxin (courtesy of Dr Grazia Isaya, Mayo Clinic).…”

“…A second immunoassay with multiplex capabilities using xMAP technology was also recently developed and validated for the measurement of frataxin levels from DBS. This assay also may be useful for population screening and monitoring purposes 24. While these immunoassays can measure significant differences between controls, carriers and FRDA patients, and they can be used for diagnostic purposes, their performance characteristics have not yet been directly compared.…”

Dried blood spots: Concepts, present status, and future perspectives in bioanalysis

“…Folate Assay for Serum folate Microbiological assay [168] 28. Frataxin Diagnosis of Friedreich ataxia (FRDA) Immunoassay [169] 29. Follicle stimulating hormone, luteinizing hormone, prolactin, testosterone, estradiol, dehydroepiandrosterone-sulfate, androstenedione, cortisol and sex hormone binding globulin Longitudinal, population-based developmental epidemiologic study of puberty [149] (Continues) [170] 31.…”