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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

Scheme of IC for detection of anti-GLA antibodies in serum.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.
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pone.0128351.g001: Scheme of IC for detection of anti-GLA antibodies in serum.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.

Mentions: For detecting anti-recombinant GLA (Aga-A or Aga-B) antibodies in serum, Aga-A or Aga-B (1 μg/line) were immobilized on the IC membrane (Fig 1, Test line; T). In contrast, anti-goat IgG antibody was immobilized on the control line (Fig 1, Control line: C) to evaluate the appropriate flow of IC by detecting goat anti-human IgG as shown in Fig 1A. In the first step, serum, which was diluted at 10 folds with sample buffer (50 mM Tris-HCl (pH = 7.2), 150 mM NaCl, 1% Trition X-100), was dropped on the IC chip (Fig 1B(1)). In the second step, the reservoir unit (Reservoir) containing conjugation buffer (50 mM Tris-HCl (pH = 7.2), 150 mM NaCl, 1% Trition X-100, 1 mM MgCl2, and AP labelled-goat anti-human IgG) was opened by fingers for developing the antibody/antigen reaction (Fig 1B(2)). The dry substrate of AP (BCIP) was fixed on the membrane and mixed with conjugation buffer after opening the Reservoir. In the final step, anti-GLA antibodies captured by the immobilized Aga-A or Aga-B on the membrane were detected by alkaline phosphatase (AP)-conjugated goat anti-human IgG, and visualized with enzyme reaction of AP (Fig 1B(3)). The level of the color strength (score) was evaluated from level 0 (no color) to level 8 (maximum density) by the visual determination according to a control color paper. We determined here that the immune reaction was positive (+) when the score was 2 or more, and pseudopositive (±) when it was one.


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)

Scheme of IC for detection of anti-GLA antibodies in serum.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128351.g001: Scheme of IC for detection of anti-GLA antibodies in serum.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.
Mentions: For detecting anti-recombinant GLA (Aga-A or Aga-B) antibodies in serum, Aga-A or Aga-B (1 μg/line) were immobilized on the IC membrane (Fig 1, Test line; T). In contrast, anti-goat IgG antibody was immobilized on the control line (Fig 1, Control line: C) to evaluate the appropriate flow of IC by detecting goat anti-human IgG as shown in Fig 1A. In the first step, serum, which was diluted at 10 folds with sample buffer (50 mM Tris-HCl (pH = 7.2), 150 mM NaCl, 1% Trition X-100), was dropped on the IC chip (Fig 1B(1)). In the second step, the reservoir unit (Reservoir) containing conjugation buffer (50 mM Tris-HCl (pH = 7.2), 150 mM NaCl, 1% Trition X-100, 1 mM MgCl2, and AP labelled-goat anti-human IgG) was opened by fingers for developing the antibody/antigen reaction (Fig 1B(2)). The dry substrate of AP (BCIP) was fixed on the membrane and mixed with conjugation buffer after opening the Reservoir. In the final step, anti-GLA antibodies captured by the immobilized Aga-A or Aga-B on the membrane were detected by alkaline phosphatase (AP)-conjugated goat anti-human IgG, and visualized with enzyme reaction of AP (Fig 1B(3)). The level of the color strength (score) was evaluated from level 0 (no color) to level 8 (maximum density) by the visual determination according to a control color paper. We determined here that the immune reaction was positive (+) when the score was 2 or more, and pseudopositive (±) when it was one.

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