Development of a Highly Sensitive Immuno-PCR Assay for the Measurement of α-Galactosidase A Protein Levels in Serum and Plasma

<div><p>Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (<i>GLA</i>) 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 <u>Mu</u>ltiple <u>S</u>imultaneous <u>Tag</u> [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.</p></div

Measurement of the GLA protein level in serum/plasma using the MUSTag beads assay.

<p><b>A.</b> The GLA concentrations in the serum samples from male patients with classic Fabry disease (Male Fabry), male subjects with p.E66Q (E66Q), and control subjects (Control). <b>B.</b> The GLA concentrations in the plasma samples collected from male patients with classic Fabry disease (Male Fabry), female patients with Fabry disease (Female Fabry), male subjects with p.E66Q (E66Q), and controls (Control). Statistically significant differences are marked with asterisks: *p<0.05; **p<0.01; ***p<0.001. All experimental data are shown as the mean (bold horizontal bars) ± S.D. (Error bars). <b>C.</b> The relationship between the enzymatic activity and protein level of GLA in the serum. <b>D.</b> The relationship between the enzymatic activity and protein level of GLA in the plasma. In each figure, closed circles (•), open circles (○), closed triangles (▴), and closed boxes (▪) show the data for samples from the Male Fabry, Female Fabry, E66Q, and Control groups, respectively.</p

Measurement of the GLA protein level in serum/plasma using ELISA.

<p><b>A.</b> The GLA concentrations in the serum samples collected from male patients with classic Fabry disease (Male Fabry), male subjects with p.E66Q (E66Q), and control subjects (Control). <b>B.</b> The GLA concentrations in the plasma samples collected from male patients with classic Fabry disease (Male Fabry), female patients with Fabry disease (Female Fabry), male subjects with p.E66Q (E66Q), and control subjects (Control). Statistically significant differences are marked with asterisks: *p<0.05; **p<0.01; ***p<0.001. All experimental data are shown as the mean (bold horizontal bars) ± S.D. (Error bars). <b>C.</b> The relationship between the enzymatic activity and protein level of GLA in the serum. <b>D.</b> The relationship between enzymatic activity and the protein level of GLA in the plasma. In each figure, closed circles (•), open circles (○), closed triangles (▴), and closed boxes (▪) show the data for samples from the Male Fabry, Female Fabry, E66Q, and Control groups, respectively.</p

Comparison of sensitivities of MUSTag plate, beads assays, and ELISA for measuring α-galactosidase A (GLA).

<p>Standard curve of the GLA protein concentration in serum (<b>A</b>) and plasma (<b>B</b>). In each figure, closed boxes (▪), closed triangles (▴), and closed circles (•) indicate the results of the MUSTag plate assay, the MUSTag beads assay, and ELISA, respectively. <b>C.</b> Sensitivities of ELISA and the MUSTag plate and beads assays for measurement of the level of GLA protein in serum (<b>C</b>) and plasma (<b>D</b>).</p

Genotype and α-galactosidase A (GLA) activity in leukocytes of patients with Fabry disease.

<p>ND: Not determined, WT: Wild-type.</p>*<p>Mean ± SD [n].</p

Structures of the immuno-polymerase chain reaction (PCR) Multiple Simultaneous Tag (MUSTag) components.

<p><b>A.</b> The MUSTag assay consists of 3 parts: a capture antibody, a recombinant protein G-avidin fusion protein, and a synthesized biotin-conjugated oligonucleotide (A). <b>B.</b> The MUSTag assay is performed similar to the sandwich enzyme-linked immunosorbent assay (ELISA) but by using a biotin-labeled detection antibody instead of an enzyme-labeled antibody. <b>C.</b> MUSTag oligo-DNAs were designed to incorporate 5 defined sequence regions: the 5′ primer for template amplification with biotin labels on sequence (F1 primer) and the complement of the 3′ primer (R primer), the 5′ primer for qRT-PCR detection (F2 primer) with R primer, an <i>Eco</i>RI restriction site, and, for hybridization with fluorogenic TaqMan, a double-labeled hybridization probe that annealed between the F2 primer and the R primer. <b>D.</b> The complement sequences of the F2 and R primers and TaqMan Probe were used for qRT-PCR measurement of the original concentration of GLA.</p