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

Nakano S, Tsukimura T, Togawa T, Ohashi T, Kobayashi M, Takayama K, Kobayashi Y, Abiko H, Satou M, Nakahata T, Warnock DG, Sakuraba H, Shibasaki F - PLoS ONE (2015)

Bottom Line: We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum.A conventional enzyme-linked immunosorbent assay supported the results.Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medical Research, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo156-8506, Japan; Synthera Technologies Co., Ltd., 4-5-1 Honkomagome, Bunkyo-ku, Tokyo 113-0021, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Characterization of ELISA and IC.A. The serum of samples #27 and #17 were assayed by ELISA for antibodies against Aga-A (black column) and Aga-B (white column) as shown in lanes 1 and 6, and also assayed after dilution of 1/20 (lane 2), 1/40 (lane 3), 1/200 (lane 4), and 1/400 (lane 5) for #27, and 1/5 (lane 7) for #17, respectively, with 0.05% Tween-20, 0.45M NaCl, 50mM sodium phosphate, pH7.4. HRP-labelled anti-human IgG antibody was applied and then washed out. The values of OD 450nm were measured after applying the HRP substrate. The data were shown as mean ± SD. 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.
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pone.0128351.g002: Characterization of ELISA and IC.A. The serum of samples #27 and #17 were assayed by ELISA for antibodies against Aga-A (black column) and Aga-B (white column) as shown in lanes 1 and 6, and also assayed after dilution of 1/20 (lane 2), 1/40 (lane 3), 1/200 (lane 4), and 1/400 (lane 5) for #27, and 1/5 (lane 7) for #17, respectively, with 0.05% Tween-20, 0.45M NaCl, 50mM sodium phosphate, pH7.4. HRP-labelled anti-human IgG antibody was applied and then washed out. The values of OD 450nm were measured after applying the HRP substrate. The data were shown as mean ± SD. 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.

Mentions: For the first evaluation of the developed ICs, we used two serum samples which had exhibited high titer of anti-GLA antibodies in the previous study; the sample #17 from a patient who had received ERT with Aga-A and #27 from one with both Aga-A and Aga-B for comparison with the results of authentic ELISA (Fig 2). We measured anti-Aga-A antibodies (black column) and anti-Aga-B antibodies (white column) for the samples at the indicated dilution by means of the ELISA (Fig 2A). In this experiment, the original serum were diluted with normal serum and used as samples. The results clearly showed the correlation to the dilution rate without background. Next, we applied the same samples to two kinds of ICs; one plotted with Aga-A (upper lane) and another with Aga-B (lower lane) (Fig 2B), and we scored each test by visual judgment according to the color scale paper (Fig 2C). Both samples without dilution clearly showed an immune reaction against both Aga-A and Aga-B. The strength of test line (T) was well correlated to the dilution rates. The sample #27 from a patient who had received ERT with Aga-A clearly indicated the cross-reaction to Aga-B.


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

Nakano S, Tsukimura T, Togawa T, Ohashi T, Kobayashi M, Takayama K, Kobayashi Y, Abiko H, Satou M, Nakahata T, Warnock DG, Sakuraba H, Shibasaki F - PLoS ONE (2015)

Characterization of ELISA and IC.A. The serum of samples #27 and #17 were assayed by ELISA for antibodies against Aga-A (black column) and Aga-B (white column) as shown in lanes 1 and 6, and also assayed after dilution of 1/20 (lane 2), 1/40 (lane 3), 1/200 (lane 4), and 1/400 (lane 5) for #27, and 1/5 (lane 7) for #17, respectively, with 0.05% Tween-20, 0.45M NaCl, 50mM sodium phosphate, pH7.4. HRP-labelled anti-human IgG antibody was applied and then washed out. The values of OD 450nm were measured after applying the HRP substrate. The data were shown as mean ± SD. 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470989&req=5

pone.0128351.g002: Characterization of ELISA and IC.A. The serum of samples #27 and #17 were assayed by ELISA for antibodies against Aga-A (black column) and Aga-B (white column) as shown in lanes 1 and 6, and also assayed after dilution of 1/20 (lane 2), 1/40 (lane 3), 1/200 (lane 4), and 1/400 (lane 5) for #27, and 1/5 (lane 7) for #17, respectively, with 0.05% Tween-20, 0.45M NaCl, 50mM sodium phosphate, pH7.4. HRP-labelled anti-human IgG antibody was applied and then washed out. The values of OD 450nm were measured after applying the HRP substrate. The data were shown as mean ± SD. 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.
Mentions: For the first evaluation of the developed ICs, we used two serum samples which had exhibited high titer of anti-GLA antibodies in the previous study; the sample #17 from a patient who had received ERT with Aga-A and #27 from one with both Aga-A and Aga-B for comparison with the results of authentic ELISA (Fig 2). We measured anti-Aga-A antibodies (black column) and anti-Aga-B antibodies (white column) for the samples at the indicated dilution by means of the ELISA (Fig 2A). In this experiment, the original serum were diluted with normal serum and used as samples. The results clearly showed the correlation to the dilution rate without background. Next, we applied the same samples to two kinds of ICs; one plotted with Aga-A (upper lane) and another with Aga-B (lower lane) (Fig 2B), and we scored each test by visual judgment according to the color scale paper (Fig 2C). Both samples without dilution clearly showed an immune reaction against both Aga-A and Aga-B. The strength of test line (T) was well correlated to the dilution rates. The sample #27 from a patient who had received ERT with Aga-A clearly indicated the cross-reaction to Aga-B.

Bottom Line: We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum.A conventional enzyme-linked immunosorbent assay supported the results.Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medical Research, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo156-8506, Japan; Synthera Technologies Co., Ltd., 4-5-1 Honkomagome, Bunkyo-ku, Tokyo 113-0021, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus