Immulon 2hb

The purified Cry6Aa was biotinylated using N-hydroxysulfosuccinimide ester-PC-biotin (Pierce, Rockford, IL) according to the manufacturer’s instructions. ELISA plates (high-binding, 96-well, Immulon 2HB; Thermo Fisher Scientific Inc., Waltham, MA) were incubated at 4°C for 12 h with 0.5 μg of ASP-1/well in 20 mM HEPES buffer (pH 8.0). The plates were then blocked at room temperature for 2 h in 100 μL PBST containing 3% BSA. For the binding assays, ELISA plates coated with ASP-1 were incubated with increasing concentrations of biotinylated Cry6Aa. For the competition assays, a 1000-fold molar excess of nonlabeled Cry6Aa was added to a solution that contained biotinylated Cry6Aa. Specific binding was determined by subtracting nonspecific binding (with 1000-fold molar excess of unlabeled Cry6Aa) from total binding (without excess unlabeled Cry6Aa). The other reaction conditions and the data analysis were conducted following the method described by Zhang et al [66 (link)]. Data were analyzed using SigmaPlot 12.0 software.

The ELISA test for antibody binding to the synthetic peptides has been carried out as already described elsewhere with minor modifications [7 (link)]. The synthetic peptides were used at a concentration of 20 μ/mL in PBS to coat polystyrene plates (Immulon 2HB, Thermo). For the detection of antirotavirus VP7 peptide IgA antibodies, only the sera whose OD readings were higher than the mean plus three standard deviations of each serum dilution of the control group were considered positive. OD values higher than 0.140 were considered positive.

The autoantibody levels were determined at 1:200 dilutions of plasma using ELISA as described previously in which 50 μg per plate of the Aβ42 peptide was coated onto microtiter wells (Immulon 2HB; Thermo, Waltham, MA) [9 (link)]. The antibodies in plasma were detected by a goat anti-mouse IgG linked to a horseradish peroxidase conjugate (Sigma; A8786) at 1:3000 dilution. Tetramethylbenzidine (TMB; Bio-Rad Laboratories Inc., Hercules, CA) was the development substrate.

Antibody levels (class IgG, and subclasses IgG1 and IgG2a) were determined by ELISA in triplicate and performed at least once as previously described [34 (link)]. Briefly, irradiated-inactivated B. pseudomallei K96243 cells (10 μg/mL in 0.1 M carbonate buffer, pH 9.5) was used to coat a 96-well Immulon 2HB, round-bottom plate (Thermo-Fisher, Pittsburgh, PA) and incubated overnight at 4 °C. After washing and blocking (1 x PBS, 1% bovine serum albumin, 0.05% Tween 20) the antigen-coated plate, two-fold dilutions of sera was made in blocking solution in triplicate, and plates incubated for 1 h at 37 °C. After washing the plate 1/5000 diluted anti-IgG- (or –IgG1 or −IgG2a) horseradish peroxidase conjugate (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) was added to the plate, and the plate was incubated for 1 h at 37 °C. The plate was washed and after color development, the plate was read at 450 nm with a reference filter of 570 nm. The results were reported as the reciprocal of the highest dilution giving a mean OD of at least 0.10, which was at least twice over the background signal ±1 SD. The results were combined with the results from the other mice in the same group, and the geometric mean with the geometric standard error of the mean of the group determined.