Ultra tmb elisa substrate

FXa in platelets was measured by a human FX specific enzyme-linked immunosorbent assay (ELISA) as reported^{10 (link)}. Briefly, a goat anti-human FX polyclonal antibody (Affinity Biologicals, Ancaster, Canada) was used as a capture antibody, and an HRP-conjugated goat anti-human FX polyclonal antibody (Affinity Biologicals) was used as a detecting antibody. The Ultra TMB-ELISA substrate (Thermo Scientific, China) was used for detection. Recombinant FXa (Haematologic Technologies, USA) was used as a standard.

Trop2 urine levels were determined by a Sandwich ELISA. Diluted anti-Trop2 capture antibody (SinoBiological, 10428-MM01, 1:100) was coated in a 96-well ELISA plate at 4 °C overnight. The wells were washed three times with PBST (PBS supplied with 0.01% Tween-20). 5% of BSA was used for blocking and incubated at 4 °C overnight. 20 µl of whole urine samples from cancer-free patients and clinically significant prostate cancer patients were incubated for 2 h at room temperature. The plates were washed three times with PBST and then incubated with the Goat-anti-Trop2-biotin detection antibody (R&D Systems; BAF650; 1:250) for 2 h at room temperature. The plates were washed three times with PBST and incubated with a streptavidin-HRP (Thermo Fisher Scientific, PI21134, 1:1000) at room temperature for 30 min. After washing with PBST three times followed by PBS two times, the signals were detected by ultra TMB-ELISA substrate (Thermo Scientific, #34028) for 20 min. After adding the stopping solution (Thermo Fisher Scientific, PIN600), plates were analyzed via plate reader (Promega) at 450 nm. The standard curve for Trop2 concentrations was determined using 0–500 pg/ml recombinant human Trop2 (SinoBiological, 10428-H08H-1).

Pleiotrophin (PTN) levels in 80 serum samples from four different patient groups (cancer-free, CPG1 and CPG5, and patients with metastatic PC) were determined by Sandwich ELISA. Diluted in 0.2 M sodium bicarbonate (pH 9.4), commercial PTN capture antibody (sc-74443, 1:250, Santa Cruz) was coated in 96-well, half-area plates (#3690, Corning) overnight at 4 °C. Then, the plates were washed three times with phosphate-buffered saline (PBS) and blocked with 5% bovine serum albumin in PBS overnight at 4 °C. Serum samples (4 μL) were incubated at room temperature for 2 h. Plates were then washed three times with PBS containing 0.01% Tween-20 (PBST) and were then incubated with biotinylated PTN detection antibody (BAF252, 1:100, R&D Systems) at room temperature for 2 h. Plates were washed three times with PBST, incubated with peroxidase-conjugated streptavidin biotin (PI21134, 1:200, Thermo Scientific) at room temperature for 1 h, washed three times with PBST and washed another two times with PBS. Bound proteins were detected using ultra TMB-ELISA substrate (#34028, as per the manufacturer’s directions, Thermo Scientific) and analysed by a Promega plate reader at 450 nm. The standard curve for PTN concentrations was determined using 0.1–200 ng/ml recombinant human PTN (252-PL-050, R&D Systems).

Aβ levels were quantified using a sensitive sandwich ELISA as previously described (Blurton‐Jones et al., 2009). In brief, immulon 2HB flat‐bottom wells (Thermo Scientific) were coated overnight at 4°C with monoclonal anti‐Aβ_1–16 (generous gift of Dr. W. van Nostrand, Stony Brook University) at 30 μg/mL in 0.1M carbonate buffer (pH 9.6). Wells were then blocked with 3% BSA in PBS at 37°C for 3 h. Next, plates were washed and wells loaded in triplicate with brain homogenates or human Aβ₄₀ or Aβ₄₂ standards (EMD Millipore) diluted in capture buffer (20 mM sodium phosphate, 0.4M NaCl, 2 mM EDTA, 0.3% BSA, 0.05% CHAPS, and 0.05% Na azide, pH 7.0) and incubated overnight at 4°C. Plates were washed again and then probed overnight at 4°C with either biotinylated monoclonal anti‐Aβ₄₀ or anti‐Aβ₄₂. After washes, wells were incubated with streptavidin‐HRP (Thermo Scientific) for 3 h at 37°C. Plates were then developed with Ultra TMB‐ELISA substrate (Thermo Scientific) followed by 0.8M Ο‐phosphoric acid to stop the reaction. Plates were then read at 450 nm using a microplate photometer (Labsystems).

Wells of microtiter plates (Greiner Bio-One, Sigma-Aldrich) were coated with 200 μL anti-rCsGSTo1 or 2 antibodies (1.0 μg/mL) in a carbonate–bicarbonate buffer (100 mM, pH 9.6) overnight at 4 °C. The plates were blocked with phosphate buffered saline containing 0.05% Tween 20 (PBS/T) and 1% bovine serum albumin (BSA) for 1 h at 37 °C, after which, 200 μL rat biliary epithelial extracts (10 μg/mL) were incubated for 2 h at 37 °C. The plates were incubated with 1:1000 diluted C. sinensis-infected rat sera (200 μL) for 1 h and subsequently with horse radish peroxidase-conjugated goat anti-rat IgG (1:2000 dilution) for 1 h at 37 °C. Color reaction was developed with Ultra TMB-ELISA substrate (200 μL/well; Thermo Fisher Scientific) in the dark for 10 min and stopped by adding 4 N H₂SO₄ (50 μL/well). Absorbance was read at 450 nm. PBS/T was used as a blank control. All results were measured after an appropriate blank correction. Each sample was assayed in triplicate.