Nunc immuno 96 microwell solid plates

For GN6 ELAA using bacterial OMVs, Nunc-Immuno 96 MicroWell solid plates (Thermo Scientific, USA) were used to immobilize bacterial OMVs in Tris buffer. After incubating OMVs at various concentrations in the plate overnight at 4 °C, the plate was washed twice and blocked using 2% BSA-Tris buffer for 2 h. After blocking, 20 pmol of GN6 aptamer and N40 control were separately added and incubated for 1 h. After washing 4 times, streptavidin-Poly HRP conjugate (Pierce, USA) was added and incubated for 30 min. After thoroughly washing 5 times in Tris buffer with 0.05% Tween-20, Ultra TMB-ELISA reagent (Thermo Scientific, USA) was added. After 15 min, 1 M sulfuric acid as stop solution was added. Absorbance at 450 nm was measured using Multiskan microplate photometer (Thermo Scientific, USA). The measured values were analyzed using non-linear regression fit model of SigmaPlot 12.0.

In total, 100 ng of recombinant AgBR1 were coated on Nunc-Immuno 96 MicroWell solid plates (Thermo Scientific) overnight at 4 °C. After being blocked with 2% non-fat milk for 1 h at 37 °C, the plates were incubated for 1 h 37 °C with serum samples serially diluted in PBS. After three washes with PBS+0.05% Tween20, the plates were incubated with horseradish peroxidase-conjugated secondary antibodies. Enzyme activity was detected by incubation with 50 μL of 3,3′,5,5′-tetramethylbenzidine solution (KPL) for 5 min at room temperature in the dark. The reaction was stopped by the addition of 50 μL of 1 M H₂SO₄. The optical density (OD) at 450 nm was measured with a microplate reader.

Similar to the Widal test, Anti-IgM and anti-IgG sandwich ELISA (MyBioSource, Inc. CA, USA) was done in all the groups. Briefly, 100 μl of coating antigen (1 μg/ml) diluted in antigen coating buffer (Immunochemistry, MN, USA) were dispensed in Nunc-Immuno 96 MicroWell solid plates (Thermo Fisher Scientific, USA) along with negative control (Only coating buffer) according to plan. The plates were incubated at 4 °C overnight and the wells were blocked using 1 % bovine serum albumin (BSA) prepared in phosphate buffer saline (PBS). The plates were washed by 125 μl washing buffer (0.1%BSA with Tween20). Hundred microliter of serially diluted sera (1:200 to 1:3200) in PBS-BSA was dispensed to each well and incubated at 25 °C for 4 h. After washing, 100 μl detector antibody conjugated with horse reddish peroxidase (HRP) was added in dilutions (1:500 anti IgA, 1:5000 anti IgG, 1:2500 anti IgM) and incubated for 30 min at 25 °C. After that, 100 μl trimethyle benzidine (TMB) substrate was added and incubated for 15 min at dark. The reaction was stopped by addition of 1 N H₂SO₄ to measure optical density (OD) at 450 nm in ELISA plate reader (Bio-Rad). Cut off values were assessed following the mean ± SD of the OD from healthy endemic controls of group D which was 0.3 for IgG and 0.2 for IgM.