Ninety-six-well immunoplates were coated with GM1-ganglioside (5 μg/mL) (Sigma-Aldrich, St Louis, Missouri, USA) and incubated at 4°C overnight. After incubation, the plates were blocked with 1% bovine serum albumin in PBS for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, a mixture of CT (2.5 ng) and serum (4 μl), which had been preincubated for 1 h at 37°C, was added to the wells and the plates were incubated for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, rabbit anti-CTB antibody (clone ab34992; Abcam, Cambridge, UK) was added to the wells and the plates were incubated for 2 h at room temperature. After washing with 0.05% Tween 20 in PBS, donkey anti-rabbit IgG conjugated horseradish peroxidase (clone Poly4046; BioLegend) was added to the wells and the plates were incubated for 1 h at room temperature. After washing with 0.05% Tween 20 in PBS, the binding of CT to GM1–ganglioside was detected by using 3,3′,5,5′-tetramethylbenzidine peroxide substrate and measuring absorbance at a wavelength of 450 nm.
Gm1 ganglioside
Manufactured by Merck Group
Sourced in United States
GM1 ganglioside is a type of glycosphingolipid found in the cell membrane. It serves as a component of the cell surface and plays a role in cellular recognition and signal transduction processes.
Automatically generated - may contain errors
Lab products found in correlation
Nunc 96 well microtiter immunoplates, by Thermo Fisher Scientific (2 mentions)
Carbonate bicarbonate buffer, by Merck Group (2 mentions)
Primary anti penta his antibody, by Qiagen (2 mentions)
Secondary antibody conjugated with hrp, by Fortis Life Sciences (2 mentions)
Hrp conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
96 well plate, by Corning (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by BD (1 mentions)
Anti ct antibody, by Merck Group (1 mentions)
Maxisorb microtiter plates, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Enzo Life Sciences (1 mentions)
Elisa reader, by BMG Labtech (1 mentions)
Tmb substrate reagent, by BD (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Goat anti rabbit igg conjugated with horseradish peroxidase, by Merck Group (1 mentions)
9 protocols using gm1 ganglioside
1
Quantitative GM1 Ganglioside Binding Assay
2
Ganglioside Receptor Binding Assay for Protein Interaction
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Ganglioside Receptor Binding Assay was performed as previously described.[54 (link)
] Briefly,
wells of the 96‐well plate (Corning) were coated overnight at 4 °C with GM1 ganglioside (Sigma). After Washed thrice with 1 × PBST, the wells were then blocked with 200 ml of PTM for 2 h at room temperature. Then, wells were washed thrice with 1 × PBST. GST or GST‐(20‐108) and/or COL1 diluted in ELISA coating buffer were coated on to the plate and incubated for 2 h at 37 °C. The plate was washed again as stated above and incubated with anti‐GST antibody (1:3000 dilution) for 1 h at 37 °C. Next, the plate was incubated with secondary HRP‐conjugated goat anti‐rabbit IgG (Santa Cruz Biotechnology). Following washing with 1× PBST, TMB substrate was added and incubated for 10 min at room temperature. The reaction was terminated by adding 50 ml of 2 N H2SO4 per well and the absorbance was read on a plate reader at 450 nm using a microplate reader.
] Briefly,
wells of the 96‐well plate (Corning) were coated overnight at 4 °C with GM1 ganglioside (Sigma). After Washed thrice with 1 × PBST, the wells were then blocked with 200 ml of PTM for 2 h at room temperature. Then, wells were washed thrice with 1 × PBST. GST or GST‐(20‐108) and/or COL1 diluted in ELISA coating buffer were coated on to the plate and incubated for 2 h at 37 °C. The plate was washed again as stated above and incubated with anti‐GST antibody (1:3000 dilution) for 1 h at 37 °C. Next, the plate was incubated with secondary HRP‐conjugated goat anti‐rabbit IgG (Santa Cruz Biotechnology). Following washing with 1× PBST, TMB substrate was added and incubated for 10 min at room temperature. The reaction was terminated by adding 50 ml of 2 N H2SO4 per well and the absorbance was read on a plate reader at 450 nm using a microplate reader.
3
GM1 Ganglioside ELISA for Recombinant Protein Detection
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The Nunc 96-well microtiter immunoplates (Thermo Fisher Scientific) were coated with 300 ng/well of GM1 ganglioside (Sigma) diluted in a carbonate-bicarbonate buffer (pH 9.6; Sigma) and incubated at 4 °C overnight. The plates were washed with TBST with 0.05% Tween 20 and blocked with 1% bovine serum albumin (BSA) in PBST for 3 h at room temperature. The recombinant protein samples were 2-fold serially diluted from 300 ng with PBST, then applied to GM1-coated wells for 4 h at room temperature. Primary anti-penta-His antibody (100 μL/well; Qiagen) and secondary antibody conjugated with HRP (100 μL/well; Bethyl Laboratories) were added for 1 h at room temperature. The plates were washed 3 times with TBST and incubated with TMB (BD Biosciences) for 30 min, and the reaction was ended upon 2N H2SO4 addition. The optical density was measured at 450 nm via an ELISA reader.
4
Quantifying Cholera Toxin in Samples
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The amount of CT in the bacterial cell supernant and fluid accumulated in rabbit ileal loop was determined by ELISA. Briefly ELISA plates were coated with 100 ng/well of GM1 ganglioside (Sigma). 100 μl of cell supernatant or intestinal fluid was used. As primary antibody rabbit polyclonal anti-CT antibody (Sigma) was used. Anti-rabbit IgG conjugated with HRP (Pierce) was used as secondary antibody. Purified CT (Sigma) was used to plot the standard curve.
5
Competitive GM1 ELISA for Anti-CT Antibodies
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Competitive GM1 ELISA was used to measure the cholera MEFA-induced (anti-CTB) antibodies against CT (B pentamer) binding to GM1. As described previously (59 (link), 60 (link)), Maxisorb microtiter plates (Nunc) coated with GM1 ganglioside (Sigma; 80 ng per well) overnight at 4°C were washed with PBST and incubated with 5% skim milk at 37°C for 1 h. After 3× washes with PBST, wells were incubated with CT (20 ng) and mouse serum dilutions (1:200–1:12,800). Washed with PBST, wells were incubated with rabbit anti-CT polyclonal antiserum (Sigma; 1:3,000), followed by HRP-conjugated goat-anti-rabbit secondary antibodies (Sigma; 1:5,000 dilution). OD650 was measured with TMB peroxidase substrate system (2-C) (KPL).
6
Quantification of Cholera Toxin Secretion
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Secretion of Cholera Toxin in culture medium was detected using a GM1-ELISA method. In short, microwell plates were coated with GM1 ganglioside (Sigma Aldrich) overnight at RT. Plates were washed with 0.05% Tween 20 in PBS buffer and blocked with 1% BSA in PBS for 30 min at RT. Supernatants from V. cholerae cultures grown to an identical optical density (OD) for ensuring equal numbers of bacteria were added in duplicate (100 μl/well) and incubated for 1 h at 37°C. For a standard curve, selected wells were supplied with purified Cholera Toxin (Enzo life sciences) in PBS with 0.1% BSA in a concentration range from 0.1 to 20 ng/ml. Immobilized CT was detected with 100 μl/well Anti-Cholera Toxin subunit B goat primary antibody (Merck Millipore), followed by rabbit anti-goat horseradish peroxidase conjugate (Jackson ImmunoResearch). Results were visualized upon addition of p-nitrophenyl phosphate buffer (Sigma-Aldrich) for conjugate activation and the resulting OD at 405 nm was measured by spectrophotometry.
7
Cholera Toxin Production Analysis
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GM1 ganglioside enzyme-linked immunosorbent cholera toxin (CT) assays were performed on 7.5-h culture supernatants for all V. cholerae strains as previously described (48 (link)). Threefold dilutions of a CT standard (List Biological Laboratories, CA, USA), uninoculated media, El Tor N16961, clinical isolate MQ1795, O395ΔtoxT, and all strains containing SNPs were prepared in GM1 ganglioside (Sigma-Aldrich, MO, USA)-coated 96-well plates. Samples were probed with anti-CTB (List Biological Laboratories, CA, USA) to determine the total nanograms of CT produced per milliliter of culture per optical density at 600 nm (OD600), and fold changes in CT production from that of N16961 were calculated.
8
GM1 Binding Assay for Pentameric Protein
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A GM1 binding assay was conducted to confirm whether samples from SEC form pentamers and to determine the specific binding affinity of samples for GM1 as the receptor for cholera toxin. Nunc 96-well microtiter immunoplates (Thermo Fisher Scientific, Rochester, NY, USA) were coated with 300 ng/well GM1 ganglioside (Sigma, Burlington, MA, USA) diluted in carbonate-bicarbonate buffer (pH 9.6; Sigma, Burlington, MA, USA) and incubated at 4 °C overnight. The plates underwent three washes with PBST (phosphate-buffered saline with 0.05% Tween 20) and were blocked with 1% BSA (bovine serum albumin) in PBST for 3 h at room temperature. The recombinant protein samples were serially diluted twofold from 300 ng first in wells with PBST and then applied to GM1-coated wells for 4 h at room temperature. Primary anti-Penta-His antibody (100 μL/well; Qiagen, Tegelen, Netherlands) and secondary antibody conjugated with HRP (100 μL/well; Bethyl Laboratories) were added for 1 h at room temperature. The plates underwent three washes with PBST and were incubated for 30 min in the dark after the addition of TMB (3, 3′, 5, 5′ tetramethylbenzidine (TMB) substrate reagent (BD Biosciences, Franklin Lakes, NJ, USA). After 30 min, 2 N H2SO4 was added, and the OD450 was measured using an enzyme-linked immunosorbent assay (ELISA) reader (BMG LABTECH, Ortenberg, Germany).
9
Evaluating LTB Protein Immunogenicity via Ganglioside Binding
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The ability of LTB protein to bind to gangliosides is indication of immunogenecity. 7, 8, 10 The microwell plate was coated with 100 μl/well of 3 μg/ml GM1 ganglioside (Sigma-Aldrich) in bicarbonate buffer, pH 9.6 at 4°C overnight. After three washes with PBST, the wells were blocked with 1% BSA in 0.01M PBS (300 µl/well) at 37°C for 2 h. The wells were washed three times with PBST and then incubated with the protein extract (100 μl/well) from the LTB transgenic carrot hairy roots for 2 h at 37°C. The wells were coated with 100 μl/well of 3.0 μg/ml BSA as a control. For the primary and secondary antibody treatments, the wells were incubated with a 1:5000 dilution of rabbit anti-LTB antibody (Sigma-Aldrich) (100 μl/well) in 0.01M PBS containing 0.5% BSA for 2 h at 37°C and washed four times with PBST. Subsequently, the wells were incubated with a 1:10000 dilution of goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma-Aldrich) (100 μl/ well) in 0.01M PBS containing 0.5% BSA for 2 h at 37°C and washed four times with PBST. Finally, the plate was incubated with100 μl/well TMB substrates (Sigma-Aldrich) for 30 min at RT in the dark. After incubation, the reaction was measured at an absorbance of 620 nm in an automated ELISA system (BioEra).
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