2 Material and Methods
2.1 Patient samples and serum preparation
All patients were recruited in the trial B-NHL 2013 (Eudra CT: 2013-003253-21) after informed consent (Table 1). NHL-BFM risk group stratification was performed as published earlier. According to protocol, treatment started with the rituximab window including one dose of rituximab 375 mg/m2 and one intrathecal triple drug administration.9 Samples were collected by the NHL-BFM study center as part of the trial B-NHL 2013 prior to the start of treatment and at day five of treatment, prior to steroid treatment. Serum was generated using serum-separating tubes. To allow blood to clot, samples were left undisturbed at RT for 20 min. To remove the clot, tubes were centrifuged for 10 min in a refrigerated centrifuge. The resulting supernatant was frozen until further use.
2.2 Rituximab ELISA
The rituximab ELISA is based on the methodology of Hampson et al., 2010. Briefly, a 96-well plate was coated with 100 µl/well of rat α-rituximab antibody (MB2A4, Bio-Rad, USA) and diluted 1:1000 in coating buffer overnight at 4°C. After blocking for 2 h at RT with 200 µl/well of 1% BSA in PBS, plates were washed three times with ELISA wash buffer. Samples were diluted 1:100000 in dilution buffer and Mabthera standard was prepared in dilution buffer at the following concentrations: 60, 50, 40, 25, 20, 10, 5, 2.5, 1, 0 ng/ml. 100 µl/well of diluted sample or standard was then added to the plate and incubated for 60 min at RT. After washing the plate five times with ELISA wash buffer, HRP-conjugated goat α-human IgG antibody (Merck, Germany) was diluted 1:60000 in blocking buffer and 100 µl/well was added to the plate, followed by an incubation of 90 min at RT. The plate was washed again five times with ELISA wash buffer and 100 µl/well of freshly prepared TMB HRP-substrate (BioLegend, USA) was added according to manufacturer’s instructions. After incubation for 45 min in the dark, the reaction was terminated by 50 µl/well of 3 M sulphuric acid. Absorbance was measured at 450 nm minus 570 nm for wavelength correction using a Synergy Mx Microplate Reader (Biotek, Germany). The standard curve was determined using a non-linear 4 parameter logistic regression (4PL) calibration model.
2.3 Staining of cells and flow cytometry
Flow cytometry measurements were performed on a CytoFLEX S flow cytometer (Beckman Coulter, Germany) and data were analyzed with the FlowJo software (version 10.0). For cell surface antigen staining, 5 µl of each human TruStain FcX and True-Stain Monocyte Blocker (Biolegend, Germany) were added to 100 µl of whole blood and incubated for 10 min at 4°C. Surface staining was performed for 30 min at 4°C by directly adding fluorochrome-conjugated antibodies diluted 1:50 or 1:100 in PBS/1% BSA. For lysis of the red blood cells, 2 ml of 1x 1-step Fix/Lyse-solution (Thermo Fisher Scientific, Germany) was added to each tube and incubated for 40 min at RT. Subsequently, samples were washed with FACS buffer. 300 µl of freshly prepared 1x Fix/Perm-buffer (Thermo Fisher Scientific, Germany) was then added to the cell pellet and incubated for 30 min at RT. After washing with 1x Perm-buffer (Thermo Fisher Scientific, Germany), intracellular staining was performed directly in the tube by adding 2 µl of each antibody. After incubation for 30 min at RT, 500 µl 1x Perm-buffer was added and samples were washed and treated with FACS buffer until measurement. The antibodies used for surface and intracellular staining are listed in Table S1.
2.4 S100A8/A9 and S100A12 ELISA
The S100A8/A9 ELISA was performed according to the manufacturers instructions (Bühlmann, Switzerland). For S100A12 serum level measurement, a 96-well plate was coated with 50 µl/well of rat α-S100A12 antibody (clone 11G7A1, Genescript biotech, USA) in coating buffer overnight at 4°C. After blocking for 1 h at RT with 100 µl/well of blocking buffer, plates were washed three times with ELISA wash buffer. Samples were diluted 1:4 in blocking buffer and S100A12 standard was prepared at the following concentrations: 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 ng/ml. 50 µl/well of diluted sample or standard was then added to the plate and incubated for 2h at RT. After washing the plate three times with ELISA wash buffer, 50 µl/well of biotinylated rat α-S100A12 (clone 3G1D5-bio, Genescript biotech, USA) was added to the plate, followed by an incubation of 30 min at 37°C. The plate was washed again three times with ELISA wash buffer and 50 µl/well of Streptavidin-peroxidase (Thermo Fisher Scientific, Germany) was added. After incubation for 30 min at 37°C and washing the plate three times, TMB substrate (Seramun Diagnostica GmbH, Germany) was added according to manufacturers instruction. After incubation for 10 min at RT in the dark, the reaction was terminated by 50 µl/well of 2N H2SO4. Absorbance was measured at 450 nm using a Synergy Mx Microplate Reader (Biotek, Germany). The standard curve was determined using a non-linear 4 parameter logistic regression (4PL) calibration model.
2.5 Statistical analysis
Data are displayed in 10–90 percentile range with median. An unpaired two-tailed student’s t-test was performed for independent sample comparisons using the GraphPad Prism software. For two-time point analysis a paired two-tailed student’s t-test was performed.P -values ≤0.05 were considered significant with p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***).