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ELISA and TLC immunostaining for detection of antibodies to ganglioside complexes
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ELISA and TLC immunostaining for detection of antibodies to ganglioside complexes

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Category
Roles of glycans during microbial infection
Protocol Name

ELISA and TLC immunostaining for detection of antibodies to ganglioside complexes

Authors
Kaida, Kenichi
Division of Neurology, Department of Internal Medicine 3, National Defense Medical College

Kusunoki, Susumu *
Department of Neurology, Kinki University School of Medicine
*To whom correspondence should be addressed.
KeyWords
Reagents

Gangliosides: purified from bovine brain

Dilution buffer: 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)

Secondary antibody: HRP (horseradish peroxidase)-conjugated goat anti-human immunoglobulin G (or M) Fc antibody

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)

H2SO4

Developing solution: chloroform, methanol, and 0.2% CaCl2·2H2O (50:45:10, v/v)

Orcinol

Resorcinol

Poly-isobutyl methacrylate

Hexane

3,3΄-diaminobenzidine tetrahydrochloride (DAB)

Coloring solution (for TLC immunostaining): phosphate-buffered saline containing 0.01% H2O2 and 50 mg/dL 3,3΄-diaminobenzidine tetrahydrochloride

Instruments

ELISA plate (96-well Polysorp flat-bottom plates)

ELISA plate reader

High performance TLC plate (Silica Gel 60, 10 × 20 cm, Merck Millipore, Billerica, MA)

TLC developing chamber

Microsyringe

Hot plate

Methods
1.

ELISA for antibodies to ganglioside complexes (GSCs)

1) 

 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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2) 

 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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3) 

 Evaporate ethanol at 37°C for several minutes and dry completely with warm air.

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4) 

 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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5) 

 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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6) 

 Remove the serum from the wells and wash three times with 300 μL of 0.1% BSA in PBS.

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7) 

 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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8) 

 Remove the secondary antibody from the wells and wash three times with 0.1% BSA in PBS.

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9) 

 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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10) 

 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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11) 

 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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2.

TLC immunostaining for antibodies to ganglioside complexes (GSCs)

1) 

 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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2) 

 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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3) 

 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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4) 

 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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5) 

 Soak the TLC plate in the coating solution for 1 min at room temperature.

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6) 

 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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7) 

 Wash the plate with 0.1 M PBS and dry it.

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8) 

 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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9) 

 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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10) 

 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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11) 

 After several to ten seconds, immunoreactants become visible due to the coloring solution.

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12) 

 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.
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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