Maxisorp 96 well microtiter plates

MaxiSorp 96-well microtiter plates (Nunc, Roskilde, Denmark) were precoated with streptavidin (1 µg/mL) for 2 h at room temperature (RT) followed by coating with biotinylated peptides (1µg/mL) for 2 h at RT. Sera diluted (1:200) in TTN were added to the microtiter plates and incubated for 1 h at RT. Patient sera were analyzed in duplicates. After careful washing with TTN buffer, AP-conjugated goat anti-human IgG diluted in TTN (1 µg/mL) was added to the wells and the plates incubated for 1 h at RT. For antibody quantification, AP activity was determined with pNPP (1 mg/mL) diluted in AP substrate buffer. The absorbance was measured at 405 nm, with background subtraction at 650 nm, using a ThermoMax microtiter plate reader (Molecular Devices, Menlo Park, CA, USA).

Following immunization, sera from all ten mice from each vaccinated or control group were collected at two-week intervals. Maxisorp 96-well microtiter plates (Nunc, Denmark) were coated with 25 μg/ml of the recombinant protein Sm29, TSP-2 or chimera (mix of chimera A and chimera B) in a carbonate-bicarbonate buffer, pH 9.6 for 16 hours at 4°C, then blocked for 2 hours at room temperature with 200 μl/well PBS-T (phosphate buffer saline, pH 7.2 with 0.05% Tween-20) plus 10% FBS (fetal bovine sera). The ELISA assay protocol was performed as described previously [19 (link)].

A homemade sandwich ELISA was used to quantify TGFBI levels in serum samples. MaxiSorp 96-well microtiter plates (Nunc, GmbH, Germany) were coated with 100 µL of anti-TGFBI antibody (clone 4G9A10; for details see Supplemental Data), at the concentration of 1 µg/mL in carbonate buffer, and incubated at 4 °C overnight. Coated wells were washed three times with PBS/Tween-20 (0.05%) and blocked with 200 µL of 10% FBS/PBS at 37 °C for 2h. After washing three times as before, 100 µL of serum sample (1:20 in PBS) was added to each well. Serial dilutions of human recombinant TGFBI (Targetome SA), from 0 µg/mL to 5 µg/mL, were prepared in PBS to serve as calibration curve. Plates were incubated at 37 °C for 1h. After washing, 100 µL of anti-TGFBI antibody (1:500 in 10% FBS/PBS; clone 4G6B10, detailed in Supplemental Data) was added to each well at 37 °C for 1h, followed, after washing, by incubation with 100 µL of anti-mouse IgG (1:3000 in 10% FBS/PBS; Dako; cat. no. P0260) at 37 °C for 1h. After washing, wells were incubated with 30% H₂O₂ and 1 mM 2,2'-Azinobis-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) solution, and the optical density was read using a Filter Max F5 plate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm.

Biotin N-hydroxysuccinimide ester and streptavidin were from Sigma-Aldrich. Goat anti-mouse IgG and IgM polyclonal antibodies were from Jackson ImmunoResearch. Rat anti-mouse IgG1, IgG2a and IgG2b antibodies were from AbD Serotec. Goat anti-mouse IgA antibodies were from CliniSciences. Sandwich ELISAs were performed with MaxiSorp 96-well microtiter plates (Nunc, Thermoscientific), and all reagents were diluted in Enzyme ImmunoAssay (EIA) buffer (0.1 M phosphate buffer [pH 7.4] containing 0.15 M NaCl, 0.1% bovine serum albumin [BSA], and 0.01% sodium azide). AEBSF (serine protease inhibitor) was from Interchim. Dialysis membranes were from Spectra/Por. Cholera Toxin and Luria Broth were from Sigma. PBS was from Gibco by Life Technologies.

The binding assays for C. albicans, C. tropicalis and S. cerevisiae enolases, and the recombinant enolases—R-Eno and R-Eno_sc, were also performed using MaxiSorp 96-well microtiter plates (Nunc) in which the human proteins were immobilized via an overnight incubation at 4 °C (3 pmol protein per well). Following this and all subsequent steps, the wells were washed three times with 1% BSA in PBS. The unoccupied surface in each well was blocked overnight with 3% BSA in PBS at 4 °C. Samples of biotinylated enolase solutions at increasing concentrations in the range of 10–500 nM (50 µL in PBS) were then added to the wells and incubated for 1.5 h at 37 °C. After washing out the unbound proteins, the binding levels were detected with the SA-HRP/TMB system.

Factor I-mediated C3b inactivation in the presence of spirochetes was assayed after pre-incubation of 1 × 10⁸ borrelial cells with PBS supplemented with 1 μg/ml FH for 1 h at RT as described (27 (link)). Following incubation, cells were washed twice with PBS and resuspended in 30 μl PBS containing 10 μg/ml C3b and 20 μg/ml factor I. To analyze C3b degradation products by Western blotting, the bacterial cells were sedimented by centrifugation and supernatant were subjected to SDS-PAGE.
In addition, cofactor activity of FH bound to purified proteins was analyzed by monitoring factor I-mediated degradation of C3b using ELISA. Briefly, purified proteins (100 ng/ml) were immobilized on Nunc MaxiSorp 96-well microtiter plates at 4°C and after blocking, wells were incubated with FH (100 ng/ml) for 1 h at RT. After washing, PBS containing C3b (10 μg/ml) and factor I (20 μg/ml) was added. Following incubation, SDS-PAGE sample buffer was added. Each reaction was loaded on a 10% Tris/tricine SDS gel and C3b inactivation products were then analyzed by Western blotting using a polyclonal anti-C3 antibody.

Following immunization, sera were collected from ten mice in each experimental group at two week intervals. The levels of specific anti-Sm10.3 antibodies were measured by indirect ELISA. Maxisorp 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with 5 µg/mL rSm10.3 in carbonate-bicarbonate buffer (pH 9.6) for 16 hat 4°C, then blocked for 2 h at room temperature with 200 µL/well PBST (phosphate buffer saline, pH 7.2 with 0.05% Tween-20) plus 10% FBS (fetal bovine serum). One hundred microliters of serum diluted 1∶100 in PBST was added to each well, and the plates were then incubated for 1 h at room temperature. Plate-bound antibodies were detected using peroxidase-conjugated anti-mouse IgG, IgG1 and IgG2a (Southern Biotechnology, CA, USA) diluted 1∶10000, 1∶5000 and 1∶2000 in PBST, respectively. The color reaction was induced by adding 100 µL of 200 pmol OPD (o-phenylenediamine) in citrate buffer (pH 5.0) plus 0.04% H₂O₂ to each well for 10 min. The color reaction was stopped by adding 50 µL of 5% sulfuric acid to each well. The plates were read at 495 nm in an ELISA plate reader (BioRad, Hercules, CA, USA). All reagents were purchased from Sigma-Aldrich, CO (St. Louis, MO, USA) unless otherwise specified.