Secondary antibody conjugated with hrp

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.

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).