Ultra tmb elisa

For ELISA-based assays, 96-well plates (Costar cat# 9018) were coated overnight at 4°C with neutravidin (Thermo Scientific, cat#31050) in 10 mM Tris pH 9 and 100 mM NaCl. After overnight incubation, non-specific binding sites were blocked with bovine serum albumin (BSA) (2% w/v) for at least 1 h at room temperature. Plates were washed in 25 mM HEPES pH 7.5, 100 mM NaCl, 0.1% BSA, and 0.01% TritonX-100 (assay buffer), and indicated biotinylated β3 peptide was incubated for at least 2 h at room temperature (0.5–3 ug/ml in di-H₂O). After washing in the assay buffer, GST-fusion proteins were added (50 ul, indicated concentrations) and incubated at room temperature for 1 h in assay buffer. For compound inhibition studies, compounds were added to GST-fusion proteins (at indicated concentrations) before they were added to plates. Plates were washed 3× with assay buffer (200 ul) to remove unbound GST-fusion protein. Horseradish peroxidase (HRP)-conjugated anti-GST antibody [Polyclonal anti-GST(Z-5)-HRP, Santa Cruz Biotechnology, cat #sc-459 0.2 ug/ml] was incubated in washed plates in 50 ul assay buffer for 45 min at room temperature. After washing, the signal was generated with 1-step Ultra TMB-ELISA (Thermo Scientific, cat#34028), stopped with 2 N H₂SO₄, and read on a plate reader (SPARK 10M/Tecan) at 450 nm.

A 96-well polystyrene high binding microplate (Corning, costar) was coated with the capture antibody (CS35 at 10μg/mL and BJ76 at 2.5μg/mL) overnight at 4°C. The plates were blocked with 1% BSA for 60 min after a brief rinse with the same. Urine spiked with LAM and the clinical samples (pretreated) were applied to the appropriate wells on the plate/s and incubated at 27°C for 90 min. After incubation, the plate/s were washed (1X PBS, Tween 80) and detection antibody (biotinylated A194-01 @ 250 ng/mL) was applied and incubated for 90 min at 27°C. The incubation was followed by washes and a 25 min incubation with streptavidin horseradish peroxidase (R&D Systems) @ 27°C). Ultra TMB-ELISA, a chromogenic substrate (Thermo Scientific) was added to the plate/s to develop the color for 30 min and the reaction was stopped by adding sulphuric acid and the optical density measured at 450nm (for the sample) and 570nm (background signal from the plate).

Maxisorp 96-well plates (Nunc) were coated overnight at 4 °C with purified 2 μg/mL of Nb484 in sodium bicarbonate buffer pH 8.2. Residual protein binding sites in the wells were blocked with 2% milk in PBS for two hours at room temperature. 50 ng of recPrP23-230, PrP^Sc (-PK) and PrP^Sc (+PK) were incubated for 2 hours with the nanobody at room temperature. Then plate was incubated with 1:2500 dilution of POM1 anti-PrP antibody[59 (link)] for 2 hours. Wells were then incubated with 1:2000 dilution of goat anti-mouse HRP (Bio-Rad) for 1 hour. Absorption at 405 nm was measured 30 min after adding 100 μL of ultra TMB-ELISA (Thermo-Scientific, Product no. 34028).

Immulon 4 HBX plates (Thermo, Milford, MA) were coated with 100 ng/well of SF162 gp140 (NIH AIDS Reagent Program) in 1x PBS and incubated at 4°C overnight. Wells were blocked with 5% milk in Tris-buffered 0.05% Tween-20 (TBST) overnight. Wells were washed and serum samples were added at 1/200 dilutions to plates and incubated at room temperature for 2 hours. Wells were washed, and immunoglobulins were detected with 1/1000 Protein A/G HRP. Wells were washed with TBST and the plates were incubated with 50 μL Ultra TMB ELISA (Thermo Fisher Scientific Inc, Rockford, IL) prior to inactivation with 50 μL 2N H2SO4. OD450 was measured on a Beckman Coulter DTX 880 Multimode Detector system.

High-binding 96-well plates (Corning) were coated with 3 μg/ml humanized mAb E16 in 100 μl coating buffer (100 mM BupH Carbonate Bicarbonate Buffer, Fisher) with pH adjusted to 9.6. Plates were washed six times with PBS containing 0.05% Tween 20 followed by incubation with 100 μl blocking buffer (PBS, 3% non-fat dry milk, and 0.05% Tween 20). RVPs were serially diluted 2-fold starting at a 1:100 dilution in 100 μl blocking buffer, added to plates, and incubated for 1 h at 37°C. Plates were washed again and were incubated with 100 μl of mouse mAb E16 diluted in blocking buffer (2 μg/ml) for 1 h at 37°C. Following washing, 100 μl of HRP-conjugated goat anti-mouse IgG (Thermo Scientific) diluted 1:10,000 in blocking buffer were added to plates and incubated for 1 h at 37°C. One-step Ultra TMB-ELISA (Thermo Scientific) substrate was added (100 μl/well) and incubated for six minutes at room temperature in the dark. The reaction was stopped by the addition of 100 μl 1N hydrocholoric acid (Fisher) and read on a plate reader (BioTek Synergy H1) at a wavelength of 450 nm.

ELISAs were performed with CN54 clade C gp140 protein from NIH AIDS Reagent Program. The antigen was diluted in phosphate-buffered saline (PBS) and incubated overnight in Immulon 4 HBX plates (Thermo) at 100 ng/well. The wells were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature (RT) for 2 h. The indicated dilutions of each sample were plated in triplicate and incubated for 3 h at RT. The wells were washed, and goat anti-mouse IgG or IgA horseradish peroxidase (Thermo Fisher Scientific Inc.) was added and incubated 2 h at RT. Wells were washed and 1 step Ultra TMB ELISA (Thermo Fisher Scientific Inc.) was added to each well. Color development was terminated by the addition of H₂SO₄. OD450 was determined on a plate reader.