Development of a highly sensitive immuno-PCR assay for the measurement of α-galactosidase A protein levels in serum and plasma.

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.

[Protein biomarker measurement and simple/rapid diagnostics with supersensitive and multiplex assay, MUSTag technology].

Recently, we face the rapid progression of an aging population, and so the importance of preventive medicine is growing. We would all like to pursue a healthy life during old age through effective treatment on the basis of the early detection of diseases. In this situation, we have developed MUSTag (Multiple Simultaneous Tag) assay technology through an innovative modification of the immuno-PCR method for the super-sensitive and multiplex detection of target biomarkers. In MUSTag technology, each different oligo-tag simultaneously detects multiplex protein targets with extremely high-level sensitivity (more than 10 fg(10(-15) g)/ml) in a dose-dependent manner by qRT-PCR (maximum: 3 plexes). Herein we report our recent results of multiple cytokine assays or disease-specific biomarker assays using MUSTag technology, and, further, clinical results from patients with cancer, ischemic brain, or heart attack, who need a prompt and predictive diagnosis for adequate treatment.

[Clinical application of supersensitive and multiplex assay, MUSTag technology].

Recently, various sets of protein biomarkers have been discovered in important diseases such as cancers, brain stroke, heart attack, diabetes, and so on. Many of these biomarkers are expected to be extremely valuable as targets for clinical diagnosis and drug development; however, the clinical validation is difficult and time-consuming by individual assays or due to very low concentration in an early stage of disease. For the super-sensitive and multiplex detection of target biomarkers, we have developed MUSTag (Multiple Simultaneous Tag) assay technology with innovative modification of the immuno-PCR method. In MUSTag technology, specific antibodies against several important biomarkers were linked to 100-300bp long oligonucleotides as detection tags. Each different oligo-tag simultaneously detects multiplex protein targets with extremely high sensitivity(more than 10 fg (10(-15) g)/ml) in a dose-dependent manner by qRT-PCR-based (maximum 3 plexes) or capillary electrophoretic amplification (over 30 plexes). Here we report our recent results of multiple cytokine assay or disease-specific biomarker assay using MUSTag technology, and further, clinical results from patients with cancers, ischemic brain or heart attack, who need prompt and predictive diagnosis for adequate treatment.