Purified single ganglioside antigens are used for enzyme-linked immunosorbent assay (ELISA) screening to identify specific antibodies associated with Guillain-Barré syndrome (GBS), an acute immune-mediated polyradiculoneuropathy, and its variants. The absence of a response to a purified single ganglioside antigen is considered to indicate an “antibody-negative” sample. However, recent studies have shown that IgG antibodies to a ganglioside complex (GSC) consisting of two different gangliosides are present in sera from patients with GBS or its variants (Kaida et al. 2004; Kaida et al. 2006), and that some “antibody-negative” sera have anti-GSC antibodies (Kaida et al. 2007).
Testing for anti-GSC antibodies may improve the detection rate of anti-ganglioside antibodies and the accuracy of diagnosis of GBS and related disorders. In turn, this may allow better prediction of prognosis and selection of therapeutic strategy. Novel glycoepitopes formed by the GSC may function as target molecules in antibody-mediated events (Kaida et al. 2010). The concept of the GSC may also influence future studies of cell adhesion and signal transduction, in which carbohydrate epitopes can play an essential role. Here, an ELISA-based method and thin-layer chromatogram (TLC) immunostaining are described for assay of anti-GSC antibodies. |
Category | Roles of glycans during microbial infection |
Protocol Name | ELISA and TLC immunostaining for detection of antibodies to ganglioside complexes |
Authors
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Kaida, Kenichi
Division of Neurology, Department of Internal Medicine 3, National Defense Medical College
Kusunoki, Susumu
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Department of Neurology, Kinki University School of Medicine
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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Gangliosides: purified from bovine brain |
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Dilution buffer: 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) |
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Secondary antibody: HRP (horseradish peroxidase)-conjugated goat anti-human immunoglobulin G (or M) Fc antibody |
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Substrate solution (for ELISA): Ortho-phenylenediamine dihydrochloride (OPD) dissolved in citrate-phosphate buffer (13 mL 0.2 M Na2HPO4, 12 mL 0.1 M citric acid, 25 mL distilled water, 20 mg OPD, and 10 μL H2O2) |
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Developing solution: chloroform, methanol, and 0.2% CaCl2·2H2O (50:45:10, v/v) |
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Poly-isobutyl methacrylate |
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3,3΄-diaminobenzidine tetrahydrochloride (DAB) |
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Coloring solution (for TLC immunostaining): phosphate-buffered saline containing 0.01% H2O2 and 50 mg/dL 3,3΄-diaminobenzidine tetrahydrochloride |
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Instruments
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ELISA plate (96-well Polysorp flat-bottom plates) |
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High performance TLC plate (Silica Gel 60, 10 × 20 cm, Merck Millipore, Billerica, MA) |
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Methods |
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ELISA for antibodies to ganglioside complexes (GSCs)
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To assay antibodies to single gangliosides, a single ganglioside dissolved in ethanol (0.2 μg/50 μL) is put in a well in the ELISA plate. |
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To assay antibodies to GSCs, two (0.1 μg each) single gangliosides are mixed in a well and left to stand for approximately 30 min. GSC antigens are prepared in a grid manner with horizontal and vertical mixing lines on the plate. |
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Evaporate ethanol at 37°C for several minutes and dry completely with warm air. |
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Add 50 μL of blocking solution (1% BSA in PBS) to each well and leave to stand for 30 min at room temperature. |
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Remove the blocking solution from the wells, add serum (primary antibody) diluted 1:40 with 1% BSA in PBS to wells (50 μL/well), and leave to stand for 90 min at room temperature. |
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Remove the serum from the wells and wash three times with 300 μL of 0.1% BSA in PBS. |
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Add HRP-conjugated secondary antibody diluted with 1% BSA in PBS to wells (50 μL/well) and leave to stand for 90 min at room temperature. |
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Remove the secondary antibody from the wells and wash three times with 0.1% BSA in PBS. |
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Add 100 μL of substrate solution (ortho-phenylenediamine dihydrochloride dissolved in 0.1 M citrate-phosphate buffer) and leave to stand at room temperature for 2 min. |
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Stop the color reaction by addition of 50 μL of 8N H2SO4. The reagent will change color from yellow to orange. |
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Read the optical density (OD) with an ELISA reader (OD, 490 nm) and correct the OD values by subtracting the OD of a well that is not coated with gangliosides (control) to obtain the antiganglioside antibody activity. |
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TLC immunostaining for antibodies to ganglioside complexes (GSCs)
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Mark lines at the spot with a pencil on a TLC plastic or glass plate. Based on the difference in development distance, the spot line for the ganglioside with the lower Rf value should be marked in a higher position on the TLC plate to ensure that the two gangliosides overlap with each other after development. |
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Apply 2 μL of the samples of single gangliosides (1 μg/μL) dissolved in chloroform/methanol solution (1/1, v/v) to the TLC plate. Each ganglioside solution is applied to an individual lane. In addition, both ganglioside solutions are applied to the neighboring lane. |
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Develop the TLC plate with a chloroform/methanol/0.2% CaCl2·2H2O (50:45:10, v/v) developing solvent in a TLC chamber, and dry the plate. |
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Prepare the coating solution (0.4% polymer): Dissolve 1.6 g of poly-isobutyl methacrylate in 64 mL of chloroform and then add hexane to make a total volume of 400 mL. The final concentration of the poly-isobutyl methacrylate solution is 0.4%. |
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Soak the TLC plate in the coating solution for 1 min at room temperature. |
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Dry the plate for more than 30 min and then put blocking solution (1% BSA in PBS) on the plate for 30 min at room temperature. |
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Wash the plate with 0.1 M PBS and dry it. |
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Put serum (primary antibody) diluted 1:100 with 1% BSA in PBS on the plate (approximately 400 μL/lane), cover the plate with parafilm, spread the primary antibody, and leave to stand for 90 min at room temperature. |
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Wash the plate with 0.1 M PBS, dry it, and then put HRP-conjugated secondary antibody diluted with 1% BSA in PBS on the plate (400 μL/lane) and leave to stand for 90 min at room temperature. |
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Remove the secondary antibody and put an appropriate amount of the coloring solution of PBS containing 0.01% H2O2 and 50 mg/dL 3,3΄-diaminobenzidine tetrahydrochloride on the plate. |
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After several to ten seconds, immunoreactants become visible due to the coloring solution. |
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Stop the color reaction by washing the plate with water. |
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Notes | ELISA
- When the corrected OD is more than 0.1, the serum sample is considered to be antiganglioside antibody-positive.
- Antibody activity to GSC (e.g. GSC A/B consisting of gangliosides A and B): Anti-A-positive or anti-B-positive sera, in which the corrected anti-A/B antibody OD is 0.2 higher than the corrected anti-A or anti-B antibody OD, are considered to be anti-A/B antibody-positive. If a serum sample has both anti-A and anti-B antibody activities, the serum is judged to be anti-A/B antibody-positive when the OD value of anti-A/B antibody is more than the sum of those of anti-A and anti-B antibodies.
- Incubation time for primary antibodies (diluted serum) can be “overnight” at 4°C.
TLC immunostaining
- TLC immunostaining is performed for gangliosides A, B, and a mixture of A and B.
- When the immunoreactivity in the lanes for A and B is weak or not observed, but strong immunoreactivity is observed in the overlapping region of A and B in the A/B lane, the sample is considered to be positive for anti-A/B antibody.
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Figure & Legends |
Figure & Legends
Figure (C) was originally published in Glycobiology. 19(7):676–92. 2009 "Antiganglioside antibodies and their pathophysiological effects on Guillain-Barré syndrome and related disorders--a review." Kaida K. et al. Oxford University Press. |
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This work is released underCreative Commons licenses
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Date of registration:2015-01-27 15:14:11 |
- Kaida, K., Morita, D., Kanzaki, M., Kamakura, K., Motoyoshi, K., Hirakawa, M., and Kusunoki, S. (2004) Ganglioside complexes as new target antigens in Guillain-Barré syndrome. Ann Neurol. 56, 567–571 [PMID : 15389898]
- Kaida, K., Kanzaki, M., Morita, D., Kamakura, K., Motoyoshi, K., Hirakawa, M., and Kusunoki, S. (2006) Anti-ganglioside complex antibodies in Miller Fisher syndrome. J Neurol Neurosurg Psychiatry 77, 1043–1046 [PMID : 16614007]
- Kaida, K., Morita, D., Kanzaki, M., Kamakura, K., Motoyoshi, K., Hirakawa, M., and Kusunoki, S. (2007) Anti-ganglioside complex antibodies associated with severe disability in GBS. J Neuroimmunol. 182, 212–218 [PMID : 17113161]
- Kaida, K., and Kusunoki, S. (2010) Antibodies to gangliosides and ganglioside complexes in Guillain-Barré syndrome and Fisher syndrome. Mini review. J Neuroimmunol. 223, 5–12 [PMID : 20172612]
- Kaida, K., Ariga, T., and Yu, R.K. (2009) Antiganglioside antibodies and their pathophysiological effects on Guillain-Barré syndrome and related disorders -a review. Glycobiology 19, 676–692 [PMID : 19240270]
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