Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

<div><p>We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.</p></div

Measurement of anti-GLA antibody levels using ELISA and IC.

<p>A. The relationship between the anti- Aga-A antibody level and anti-Aga-B antibody one measured by means of ELISA. B. The relationship between IC and ELISA for anti-GLA antibody level using Aga-A. C. The relationship between IC and ELISA for anti-GLA antibody level using Aga-B. In each figure, closed rhombuses (♦), closed boxes (■), closed triangles (▲), and crosses (×) show the data for samples from Fabry patients treated with Aga-A, Aga-B, and the both, and control subjects, respectively. Control exhibits the average value of the data of healthy people (n = 20).</p

Measurement of serum anti-GLA antibodies in Fabry patients received ERT and their phenotype and genotype.

<p>¹⁾Aga-A; agalsidase alpha</p><p>²⁾Aga-B; agalsidase beta</p><p>³⁾IC; immunochromatography</p><p>⁴⁾OD; optical density at 450nm</p><p>⁵⁾Score: 8 scale-values of line density by visual measurement</p><p>⁶⁾ERT; enzyme replacement therapy</p><p>⁷⁾NA; not available</p><p>⁸⁾WT: wild type. Each serum was diluted 100 folds and 5 folds with healthy control serum before ELISA and IC, respectively.</p><p>⁸⁾Cont: average values of healthy control (Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128351#pone.0128351.s006" target="_blank">S1 Table</a>).</p><p>Measurement of serum anti-GLA antibodies in Fabry patients received ERT and their phenotype and genotype.</p

Comparison of anti-GLA antibody levels in clinical samples using ELISA and IC.

<p>Sera from 29 Fabry patients (#1–29) and controls (Cont) were assayed by ELISA (left side) and IC (right side) as refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128351#pone.0128351.t001" target="_blank">Table 1</a>. Control exhibits the average value of the data of healthy people (n = 20) used blank ELISA without both Aga-A neither Aga-B. According to the protocol, ELISA was performed using magnetic beads attached with Aga-A or Aga-B as antigens and the anti-GLA antibodies in each 100-fold diluted serum were measured with colorimetric methods of OD 450 nm using AP-labelled-anti-human IgG antibody and the substrate. The same serum were 5-fold diluted and applied to IC for Aga-A (black column) and Aga-B (white column), and judged by the color scale. The all data were shown as mean ± SD.</p

Scheme of IC for detection of anti-GLA antibodies in serum.

<p>A. Scheme of an IC chip which was designated for detecting anti-GLA antibodies in serum. Sample is dropped into the middle hole (arrow) and the reaction buffer including AP-labelled anti-human IgG in the reservoir unit is running on the membrane after a break by finger. C means control line and T does test line. B (1). The principle of IC for Aga-A and Aga-B. Mixture solution of serum and buffer including anti-GLA antibodies from serum of Fabry patients who received ERT with Aga-A and/or Aga-B was applied into the sample hole, and then the solution is running in the nitrocellulose membrane. B (2). After breaking the reservoir unit (Reservoir), the reaction buffer including AP-labelled anti-human IgG and substrate for AP which was stored in separate space in the reservoir unit is running on the membrane. B (3). If the sample has anti-GLA antibodies, the detection antibody on the membrane of IC captures the antibody/AP-labelled anti-human antibody/AP-substrate complex, and blue line appears on the test line (T). On the control line. The AP-labelled anti-human IgG antibody is trapped and makes blue color line, showing that the system works well (C). B. The same samples were applied to IC for Aga-A (upper lane) and Aga-B (lower lane), and the color line was scored from 0 to 8 after 15 min later. The color scale is shown in C.</p