Emax plate reader

96-well ELISA plates (Costar) were coated with 50 μl of purified antibodies at 5 μg/ml in 50 mM sodium carbonate buffer, pH 9.5, 50 μl/well for 2 hrs at 37°C, and blocked with 3% BSA for 90 min at 37°C. His tagged TRAP or TRAP-CSP fusion proteins (50 μl, 0.4 μg/ml) was added and incubated at 4°C overnight. Binding was detected with HRP-anti-His (Penta-His Ab at 1:5000 dilution) or with biotin-labeled primary antibody at 0.5 μg/ml followed by HRP-streptavidin. 10 min after addition of peroxidase substrate (Life Technologies), plates were read at 405 nm on an Emax plate reader (Molecular Devices).

Microtiter plates (NUNC Maxi-Sorp, Rochester, NY, USA) were coated with a rabbit anti-rhuApo2L/TRAIL polyclonal antibody, and then incubated at 2–8 °C from 12 to 72 h. After the plates were blocked, standards, serum samples and controls were added to the plate and incubated for 1–2 h. Captured rhuApo2L/TRAIL was detected with a biotinylated monoclonal antibody (Mab 5C2). Next, strepavidin horseradish peroxidase (Southern Biotech, Birmingham, AL, USA) was added. For color development, tetramethyl benzidine peroxidase substrate (two-step TMB; KPL, Inc., Gaithersburg, MD, USA) was added. Lastly, 1 M phosphoric acid was added to stop the reaction and plates were read at an absorbance of 450 nm (Molecular Devices Emax plate reader, Sunnyvale, CA, USA). The minimum quantifiable concentration in this assay was 62.5 ng/ml. Accuracy, intra-assay precision and inter-assay precision were acceptable during the validation experiments.

Plasma samples were assayed by PEPSCAN analysis using SIV_mac251 gp120 linear peptides^{16 (link)}. ELISA plates (Nunc Maxisorp) were coated with 100 ng of each of the 1–89 overlapping peptides (with 15 amino acids each encompassing the entire SIV_mac251 gp120 sequence) in 50 mM NaHCO3, pH 9.6, per well, incubated overnight at 4 °C, and blocked with 200 μl of Pierce SuperBlock blocking buffer in PBS for 1 h at room temperature. Serum samples were diluted at 1:50 in sample diluent (Avioq), and 100 μl were added to the plate and incubated for 1 h at 37 °C. Plates were washed six times with PBS Tween 20 (0.05%) and incubated with 100 μl anti-human HRP diluted at 1:120,000 in sample diluent (Avioq) to all wells and incubated, covered, for 1 h at 37 °C. The plates were again washed six times and developed using 100 μl of K-Blue Aqueous substrate (Neogen) to all wells and incubated 30 min at room temperature. The reaction was stopped by adding 100 μl of 2 N sulfuric acid to all wells and plate was read at 450 nm on a Molecular Devices E-max plate reader.

To quantify circulating blood plasma levels of RNASET2, an ELISA was developed (Supplemental Figure S6) with detection range of 100pg/ml-2ng/ml (Supplemental Figure S6D). High binding ELISA plates were coated overnight with 50μl of 1/100 diluted Rabbit anti-RNASET2 (cat. 041159 US biological Salem, MA). Plates were washed and samples and standards were added for 24h followed by addition of 50ul of 1/300 diluted polyclonal mouse anti-RNASET2 (cat. H00008635-B01P Novus Biological Littleton, CO) for 2h. This was followed by washing and addition of 100μl of 1/2000 diluted alkaline phosphatase-conjugated goat anti mouse (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. Substrate, 0.2 mM NADP (Sigma-Aldrich, St. Louis, MO) was added for 30 min followed by addition of amplifier (3% 2-propanol, 1 mM iodonitrotetrazolium violet, 75μg/ml alcohol dehydrogenase, and 50μg/ml diaphorase; Sigma-Aldrich) for 10 min. Plates were read at 490 nm using an E max plate reader (Molecular Devices, Sunnyvale, CA).