Primary NK cells were incubated on polystyrene flat-bottomed 96-well plate (Nunc) coated with MICA (R&D Systems) or rituximab (GlaxoSmithKline), both with ICAM-1 (R&D Systems) or ICAM-1 alone in binding buffer (carbonate bicarbonate; Sigma-Aldrich) at 37°C for 18 h. Cell supernatants were collected and centrifuged at 350 g for 10 min at 4°C to remove cell debris. IFN-γ secretion was quantified from the supernatants by sandwich ELISA. For this, ELISA plates were coated with anti–IFN-γ mAb (1 µg/ml; clone NIB42; BD) in binding buffer (carbonate bicarbonate; Sigma-Aldrich) and blocked with 1% BSA/0.05% Tween-20/PBS. Supernatants were added to the plate and incubated for 1 h at RT. Plates were washed and incubated with biotinylated anti–IFN-γ mAb (1 µg/ml; clone 4S.B3; BD) and then streptavidin HRP (BD). The plates were developed with TMB ELISA substrate (Sigma-Aldrich), and the reaction was stopped with 1 N H2SO4. Absorbance was measured at 450 nm using a 570-nm reference line to compensate for optical interference.
Tmb elisa substrate
Manufactured by Merck Group
Sourced in United States
TMB ELISA substrate is a chromogenic substrate used in enzyme-linked immunosorbent assay (ELISA) procedures. It is a tetramethylbenzidine (TMB) solution that produces a blue color change when oxidized by the horseradish peroxidase (HRP) enzyme. The intensity of the color change is proportional to the amount of target analyte present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Duoset elisa, by R&D Systems (2 mentions)
Rituximab, by GlaxoSmithKline (1 mentions)
Icam 1, by Merck Group (1 mentions)
Icam 1, by R&D Systems (1 mentions)
Streptavidin hrp, by BD (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Cp004, by R&D Systems (1 mentions)
Control aptamer, by Integrated DNA Technologies (1 mentions)
Hrp conjugated anti dig antibody, by Roche (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
6 protocols using tmb elisa substrate
1
Quantification of IFN-γ secretion from primary NK cells
2
Cytokine Secretion Assay for NK Cells
3
Pancreatic MPO Activity Assay
4
Assessing Trastuzumab's Fc-Mediated C1q Binding
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The C1q binding assay was performed to assess Fc-related biological activity of trastuzumab using sandwich enzyme-linked immunosorbent assay (ELISA). In this system, 96-well plates were coated with seven concentrations (0.391–25 μg/mL) of trastuzumab and incubated at 37 °C for 1 h. Then, the plate was blocked using blocking buffer and incubated at room temperature for 1 h. After blocking, a fixed concentration of C1q (Quidel) solution was added to the plate and the plate was incubated at room temperature. After incubation, fixed concentration of C1q-HRP antibody (Abcam) was added to the plate and the plate was incubated at room temperature. Using 3,3′,5,5′-tetramethylbenzidine (TMB) ELISA substrate (Sigma-Aldrich, St. Louis, MO, USA), C1q binding activity was quantified by measuring the absorbance at 450 nm using a SpectraMax® M3 (Molecular Devices). Data were analyzed by using PLA software to calculate the relative binding activity.
5
Quantification of M-CSF production
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Primary monocyte-derived macrophages were incubated on chambered glass coverslips coated with PLL, human CD47-Fc, or human IgG, as indicated, at 37°C for 24 h. Cell supernatants were recovered and centrifuged at 350 g for 10 min at RT to remove cell debris. M-CSF production was quantified in the supernatants by sandwich ELISA (DuoSet ELISA; R&D Systems), according to manufacturer’s instructions. The plates were developed with TMB ELISA substrate (Sigma-Aldrich), and the reaction was stopped with 1 N H2SO4. Absorbance was measured at 450 nm using a 570-nm reference line.
6
Quantitative Aptamer-Based Protein Detection
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Streptavidin‐coated 96‐well ELISA plates (R&D systems, CP004) were washed twice in PBS‐T (PBS 1× + 0.05% Tween‐20), then incubated for 1 h at room temperature with 0.025 μM Biotinylated peptides, blocked by incubation with 1% BSA (Sigma, A9647) in PBS for 30 min at 37°C, and then incubated with 0.04 μM digoxigenin (3′‐DIG) AS1411 or control aptamer (Integrated DNA Technologies) in PBS for 1 h at room temperature. Then, plates were incubated with HRP‐conjugated anti‐DIG antibody (Roche, 11207733910) diluted 1/2,000 in 1% BSA‐PBS for 30 min at 37°C. Signal was detected using TMB ELISA substrate (Sigma, T0440). Plates were washed three times before each subsequent step with PBS‐T and five times prior to signal detection. Signal was read using a Tecan5 plate reader reading absorbance at 360 nm wavelength.
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