Enzyme-linked immunosorbent assay (ELISA) was used to detect rotavirus antigens in mouse feces, as previously described.3 (link),6 (link) Briefly, 96 well EIA/RIA plates (Costar, 3590) were coated with Rabbit Anti-rotavirus Group-A (Biorad, AHP1360) capture antibody overnight at room temperature and blocked with 200ul of 1% BSA. Mouse fecal homogenates were prepared at 100 mg/mL concentration and were centrifuged to remove all debris. After blocking, supernatants of the homogenates were then incubated in the blocked plates. Stock murine rotavirus was used as a control. Hyperimmune Guinea pig anti-RRV were diluted at 1:1000 in 1% BSA and used as detection antibody. Followed by the incubation of HRP Donkey Anti-Guinea Pig antibody (Jackson ImmunoResearch, cat#706-035-148) secondary antibody at 1:5000 dilution in 1% BSA. All incubation steps after capture antibody were at 1 h at room temperature. TMB ELISA Substrate Solution (Invitrogen, cat#00420156) was utilized to develop the signal. TMB stop solution (KPL, cat# 50–85-04) was added after 5 minutes of TMB incubation. OD readings were taken at 450 nm.
Tmb elisa substrate solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
TMB ELISA substrate solution is a liquid reagent used in enzyme-linked immunosorbent assay (ELISA) protocols. It serves as a chromogenic substrate that undergoes a color change reaction when exposed to the enzyme-labeled antibodies or antigens in the ELISA assay. The color change can be measured spectrophotometrically to quantify the target analyte.
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Lab products found in correlation
Fluostar omega microplate reader, by BMG Labtech (3 mentions)
Eia ria plate, by Corning (2 mentions)
Hrp donkey anti guinea pig antibody, by Jackson ImmunoResearch (2 mentions)
Costar 9018, by Corning (2 mentions)
D3664 2mg, by Merck Group (2 mentions)
Dtx 880 plate reader, by Beckman Coulter (2 mentions)
Prism v 6.0b software, by GraphPad (2 mentions)
A synthetic myc epitope peptide, by AnaSpec (2 mentions)
Scl 10a hplc, by Shimadzu (2 mentions)
Anti mouse igm hrp, by Merck Group (2 mentions)
A8786, by Merck Group (2 mentions)
Dna sodium salt from calf thymus, by Merck Group (1 mentions)
Versamax 190 microplate reader, by Molecular Devices (1 mentions)
Goat anti rat hrp, by Santa Cruz Biotechnology (1 mentions)
Donkey anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions)
Maxisorp flat bottom 96 well plate, by Thermo Fisher Scientific (1 mentions)
Cyan adp lx, by FlowJo (1 mentions)
Vivotag 645, by PerkinElmer (1 mentions)
Live dead violet stain, by Thermo Fisher Scientific (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
21 protocols using tmb elisa substrate solution
1
Rotavirus Antigen Detection in Mouse Feces
2
Rotavirus Detection in Mouse Feces
3
Enzyme-Linked Immunosorbent Assay (ELISA) Protocol
4
Optimized ELISA Protocol for Antibody Detection
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ELISAs were performed as reported previously [27 (link)]. Briefly, 96-well plates (Costar 9018, Corning Incorporated) were treated with poly-L-lysine solution for 2 hours and then coated with DNA sodium salt from calf thymus (D3664-2MG, Sigma-Aldrich). Sera samples (three 1:2 serial dilutions), and HRP-labeled secondary antibodies were diluted in a 1.5% BSA-PBS. Secondary antibodies against mouse IgM (1020-05), IgG (1031-05), IgG1 (1070-05), IgG2a (1080-05), IgG2b (1091-05), and IgG3 (1101-05) were obtained from Southern Biotech (Birmingham, AL, USA). To develop the ELISA we used 100 μL of 1X TMB ELISA substrate solution (eBioscience 00-4201-56, San Diego, CA, USA) per well and incubated 10 minutes in the dark at RT. The reaction was stopped using 50 μL of 2N H2SO4, read and analyzed in a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).
5
Quantifying Drosophila Insulin-Like Peptides
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Twenty 7–11-day old males were decapitated and placed in a 0.2 mL PCR tube with a hole punctured in the bottom. The tube was placed into another collection tube and centrifuged at 5000 rpm for 3 min at 4 °C. The extracted hemolymph was diluted with 100 μL cold PBS. 45 μL of the diluted hemolymph was coated on a MaxiSorp flat-bottom 96 well plate (Nunc) overnight at room temperature. The plate was incubated in block (0.02M NaPO4 buffer pH 7.4, 150 mM NaCl, 1.27 mM EDTA, 1% BSA) for 1 hour at room temperature. The plate was washed twice with 0.05% Tween-20 in PBS then incubated with either rat anti-dILP2 (1:1000) or rabbit anti-dILP5 (1:2000) for 2 hours at room temperature. After three washes the plate was incubated with goat anti-rat HRP (1:2500, Santa Cruz) or donkey anti-rabbit HRP (1:2500, Santa Cruz) secondary antibody for 1 hour at room temperature. The plate was washed three times then incubated with 1X TMB ELISA substrate solution (eBioscience) for 15 min. The reaction was stopped with 1M phosphoric acid and the absorbance at 450 nm was measured using a VersaMax 190 Microplate Reader (Molecular Devices).
dILP2 and dILP5 levels were normalized to protein levels measured by BCA Protein Assay (Pierce). Anti-dILP2 and anti-dILP5 antibodies were provided by P. Leopold [26] (link).
dILP2 and dILP5 levels were normalized to protein levels measured by BCA Protein Assay (Pierce). Anti-dILP2 and anti-dILP5 antibodies were provided by P. Leopold [26] (link).
6
Phage Binding Quantification by ELISA and Flow Cytometry
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1-step ultra tetramethylbenzidine (TMB)-ELISA substrate solution (ThermoFisher Scientific, Middletown, VA) was allowed to warm to room temperature. Cells were prepared as described in cell preparation. Cells were washed with DPBS+/1%BSA, 100 uL per well. For each type of phage and control wild-type KE phage, three wells were incubated with 40 uL of the phage (10e8 phages/uL). Phage were incubated on cells for 1 h in a humidified, 37°C, 5% CO2 incubator. Cells were then washed three times (DPBS+/1%BSA, 100 uL) and fixed (100 uL of 2% PFA, 5 min), then washed twice more. HRP-αM13 pIII monoclonal antibody (100 uL, 1:3000 dilution in DPBS+/1% BSA, NEB) was added to each well for 1 h. Cells were washed four more times and 100 uL of TMB was added. After the TMB reacted with the HRP, the absorption was measured on a microplate reader at 650 nm. Flow cytometry was performed by binding fluorescently labeled phage (Vivotag 645, PerkinElmer, Waltham, MA [31 (link)]) to CAF or MRC5 for 30 min. Cells were washed three times and live-dead violet stain (Life Technologies) was added. Data was gathered on Beckman Coulter CyAN ADP LX and gated on cell population, live cells, and phage positive cells using FlowJo software.
7
Pharmacokinetic Profiles of Bi-PB-1.2 Antagonists
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Example 12
To evaluate the pharmacokinetic properties of Bi-PB-1.2C and Bi-PB-1.2D relative to Bi-PB-1.2 (wt) in vivo, pharmacokinetic profiles were generated. Briefly, 10 mg/kg of each antagonist was intravenously injected into the tail vein of 6-10 week old female CD1 mice (n=2 mice per molecule). Serum was harvested at 3 minutes, 3 hours, 1 day, 3 days, 7 days and 10 days post injection. To detect the antibodies in the serum, 96 well ELISA plates were coated with 5 μg/ml goat anti-human IgG F(ab′)2 fragment (Sigma, # SAB3701274) and then blocked with 5% milk in PBS. Serially diluted mouse serum in 5% milk and serially diluted purified protein molecule as standard were added to the plates. Following incubation with Peroxidase AffiniPure Mouse Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, #209-035-098) and further washes, Bi-PB-1.2 (
8
Quantification of HuNoV Structural Proteins
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HuNoV-infected Vero cells were freeze-thawed 2× then the supernatants were transferred to high binding ELISA plates (Corning, Corning, NY, USA) and incubated at 4 °C overnight. The plates were washed 3× with PBS before blocking 2× with SuperBlock (Thermo Fisher Scientific) for 15 min at RT. Following the incubation, a 1:1000 dilution of either a polyclonal rabbit IgG anti-VP1 antibody (Abcam, Cambridge, UK) or a polyclonal rabbit IgG anti-p48 antibody (gift from Christiane Wobus) in SuperBlock was added to the wells for 1 h at 37 °C. After removal of the solution, the wells were washed 3× with KPL wash buffer and a 1:1000 dilution of HRP-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) in SuperBlock was added to the wells and incubated at 37 °C for 1 h. The solution was decanted and the cells were washed 3× with KPL wash buffer and a TMB ELISA Substrate Solution (Thermo Fisher Scientific) was added for colorimetric visualization. OD450 was read using an Epoch Microplate Spectrophotometer (Biotek).
9
Protein Expression Analysis in UV-Irradiated NHEK-Ad Cells
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NHEK-Ad cells (105) were plated on 6-well cell-culture plates (SPL). At 80% confluence, the wells were pre-treated with DeinoPol (10 μg) followed by irradiation with 120 mJ/cm2 UV light. After 6 h of incubation at 37 °C, cells were lysed with RIPA buffer (Sigma-Aldrich) and separated on Bis-Tris Bolt gel (Invitrogen; Carlsbad, CA, USA) followed by transfer onto nitrocellulose membranes (Bio-Rad; Hercules, CA, USA). Membranes were blocked in 5% skimmed dry milk (Bio-Rad) in 0.05% Tween-20 in PBS (PBS-T) and then incubated with anti-Bcl-2 IgG1 monoclonal antibody (Santa Cruz Biotechnology; Santa Cruz, CA) or anti-Bax IgG2b monoclonal antibody (Santa Cruz Biotechnology). The membrane was then washed and incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin (Southern Biotech; Birmingham, AL, USA). Membrane-bound peroxidase was detected by TMB-ELISA substrate solution (Thermo-Fisher; Waltham, MA, USA).
10
NP-Specific Antibody Response Evaluation
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Blood serum was collected prior to surgery and NP vaccine injection, and at 0, 2, 4, 8 and 12 weeks after these procedures to detect the presence of anti-NP antibody responses using an NP ELISA custom created for this study. Briefly, a 96-well Immulon plate (ThermoFisher) was coated with 100 μL of NP protein isolated from pooled sheep NP material collected at necropsy. The NP protein was diluted to a concentration of 20ug/mL in carbonate buffer. After incubation overnight at 4°C, wells were washed with PBS Tween using a plate washer, and non-specific binding sites were blocked for 2 h at room temperature with PBS + 10% BSA. After washing with PBS, 100 μL of rabbit serum (diluted 1:100 in 1%BSA/PBS) were added to the plates and incubated for 2 h at room temperature. After a second plate wash, 100 μL of 1:3000 dilution (in PBS + 1% BSA) of peroxidase conjugated donkey anti rabbit IgG (Jackson Immuno Research) was added and incubated for 1 h at room temperature. Following washing with PBS, TMB-ELISA Substrate Solution (ThermoFisher) was added and incubated for 10 min. Finally, 50 μL TMB stop solution was added and the optical density (OD) was measured at an absorbance at 450 nm. Optical density values were plotted, and pre-vaccination serum ODs were compared to post-vaccination ODs.
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