Tumour-specific immune responses to IFN-γ were measured in vitro by ELISPOT assay (Mouse IFN gamma ELISPOT Ready-SET-Go!; Cat. No. 88-7384-88; eBioscience). A Millipore Multiscreen HTS-IP plate was coated overnight at 4 °C with anti-Mouse IFN-γ capture antibody. Single-cell suspensions of splenocytes were obtained from MC38 tumour-carrying mice and seeded onto the antibody-coated plate at a concentration of 2 × 105 cells per well. Cells were incubated with or without KSPWFTTL stimulation (10 μg ml−1; in purity >95%; PEPTIDE 2.0) for 48 h at 37 °C and then discarded. The plate was then incubated with biotin-conjugated anti-IFN-γ detection antibody at r.t. for 2 h, followed by incubation with Avidin-HRP for 2 h at r.t. 3-amino-9-ethylcarbazole substrate solution (Sigma, Cat. AEC101) was added for cytokine spot detection.
Avidin hrp
Manufactured by Merck Group
Sourced in United States
Avidin-HRP is a laboratory reagent that consists of the protein avidin conjugated to the enzyme horseradish peroxidase (HRP). Avidin has a high affinity for the small molecule biotin, and the HRP enzyme can catalyze colorimetric or chemiluminescent reactions. This product is commonly used in various bioassays and detection methods that involve biotin-avidin interactions.
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Lab products found in correlation
Multiscreen hts, by Merck Group (1 mentions)
Tcs sp5 laser scanning microscope, by Leica (1 mentions)
Biotinylated anti mouse igg, by GE Healthcare (1 mentions)
Las af software, by Leica (1 mentions)
Avidin cy 2, by Jackson ImmunoResearch (1 mentions)
Elispot image analyzer, by Cellular Technology (1 mentions)
Tetramethylbenzidine tmb substrate, by BD (1 mentions)
Anti inos antibody, by Merck Group (1 mentions)
Biotinylated anti rabbit igg secondary antibody, by Vector Laboratories (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
96 well microtiter plate, by Corning (1 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Avidin agarose, by Merck Group (1 mentions)
Escherichia coli bl21 cells, by Promega (1 mentions)
Trypsin, by Promega (1 mentions)
10 protocols using avidin hrp
1
Measuring Tumor-Specific IFN-γ Responses
2
Neurofascin Immunostaining Protocol for Confocal Microscopy
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For confocal microscopy, anti-neurofascin antibody was applied at a dilution of 1∶100 at 4°C in a wet chamber over night. After washing with phosphate buffered saline (PBS) biotinylated anti-mouse IgG (GE Healthcare, Buckinghamshire, UK) was applied for 1 hour at room temperature at a dilution of 1∶200, followed by Avidin HRP (1∶100; Sigma, St. Louis, USA) for one 1 hour. After enhancement with 0,1% CSA in PBS/0.03% H2O2 for 20 min at room temperature (protocol as described in [30] (link)) signal was visualized by Avidin-Cy2 (Jackson ImmunoResearch, West Grove, PA, USA) at 1∶75 for one hour at room temperature. For detection of bound anti-neurofascin antibody in fetal tissue slides were subjected to the protocol as described above but without applying the anti- neurofascin antibody in the first step. Fluorescent preparations were examined using a Leica TCS SP5 laser scanning microscope with LAS AF software (Leica Microsystems, CMS GmbH, Germany). An argon laser was used for detection of Cy2 (488 nm excitation) signal.
3
Quantification of Allosensitized T Cells
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ELISPOT assay was used to analyze the numbers of IFNγ-producing T cells allosensitized by the direct and indirect pathway, as described previously19 (link). In brief, 96-well ELISPOT plates (Multiscreen, Darmstadt, Germany) were coated with anti-IFN- antibody (BD Pharmingen, San Diego, CA), and blocked with 1% BSA. Purified T cells from allografted BALB/c mice two weeks post-transplantation (250,000 CD4+ T cells, MACS-sorted) were incubated with (1) C57BL/6 APCs (500,000 CD90.2−, MACS sorted splenocytes) to quantify frequencies of directly allosensitized T cells or (2) with syngeneic APCs pulsed with sonicated donor antigen (from 106 cell/50 µl single cell suspension of C57BL/6 spleen) to quantify frequencies of indirectly allosensitized T cells. T cells harvested from LNs of naive BALB/c animals served as controls. After 48 hours of incubation, plates were washed and a biotinylated anti-IFN-γ detection mAb was added at 2 μg/ml (BD Pharmingen, San Jose, CA) and incubated overnight at 4 °C. The plates were then washed, incubated for 1 hour with Avidin-HRP and developed using aminoethylcarbazole staining solution (Sigma-Aldrich, Springfield, MO). The resulting spots were analyzed using the computer-assisted ELISPOT image analyzer (Cellular Technology Ltd., Cleveland, OH).
4
Quantitative Measurement of iNOS
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The iNOS contents were determined using the method described previously by Marshall et al. [29 (link)] and modified by Saia et al. [18 (link)]. The macrophage homogenates were diluted in phosphate-buffered saline (PBS 0.1 M, pH 7.2) and incubated overnight at 4°C in Nunc Maxisorb plates (Life Technologies, Paisley, UK). The wells were washed with PBS containing 0.05% Tween-20 (PBS-T) and blocked with 1% (w/v) bovine serum albumin (BSA) for 1 h at room temperature. Subsequently, the polyclonal rabbit anti-iNOS antibody (1:1000; Sigma) was added for 1 h at room temperature. After washing, the biotinylated anti-rabbit IgG secondary antibody (1:200; Vector Laboratories) was added to each well, incubated with avidin-HRP (1:5000; Sigma) for 30 min, and the colour developed by addition of 3,3′,5,5′-tetramethylbenzidine substrate (TMB; BD Biosciences, Franklin Lakes, NJ, USA). The reaction was stopped with 2 N H2SO4 and the optical density (OD) reading at 450 nm was taken. The sample levels of iNOS were expressed as OD normalized to the respective total protein concentrations.
5
ELISA for Anti-RBD Antibody Detection
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Direct ELISA for detecting anti-RBD antibodies was performed from day 7 or day 14 sera samples as previously described12 (link),47 (link). Briefly, WT-RBD protein (2 µg/ml) was coated overnight in coating buffer in 96 well high-affinity ELISA plates (Nunc) at 4 °C. Next day, 5 % skimmed milk (blocking buffer) was used for blocking and then sera samples from the infected mice were added to the RBD bound ELISA plates at different dilutions and incubated at room temperature (RT) for 1 h. Next, three washing steps with wash buffer (PBS + 0.05 % tween 20) were performed followed by incubation with anti-mouse IgG antibody detection antibody conjugated with biotinylated (1:1000 dilution) for 1 h. The wells were subsequently washed and incubated further with the enzyme Avidin-HRP (1:10000 dilution) (Sigma) for 30 min at RT. Finally, wells were washed five times and TMB substrate (Thermo Fisher Scientific) 50 µl was incubated in the dark for 15–20 min at RT. The enzyme reaction was stopped by adding 50 µl of 1 N H2SO4. OD was recorded at 450 nm in a microtiter plate reader.
6
Phage Capture ELISA for Detecting Protein Interactions
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Phage capture ELISAs were performed essentially as described previously [38 (link)]. Briefly, unconcentrated supernatant containing the scFvs or Ni–NTA purified scFvs was added to the wells of high binding ELISA plates for 1 h at 37 °C on a shaker. All incubations were performed at 37 °C whilst shaking. After washing with 0.1% PBST three times, the wells were blocked with 2% milk in PBS. Next, human brain tissue (2–10 μg/ml) or sera (1/100 v/v) was added followed by 200 ng/ml of the 40 mmol carboxyl biotinylated phage. After addition of the secondary antibody avidin-HRP (Sigma-Aldrich, USA), enzyme detection was again achieved using the SuperSignal ELISA Femto Maximum Sensitivity Substrate kit.
When screening the potential detection phage using the capture ELISA system, the phage was not biotinylated and therefore detected using the anti-M13 HRP antibody. Similarly, any reactivity with the commercial anti-TDP-43 antibody was recognized with the goat anti-rabbit IgG HRP secondary. Lastly, the quantity of immunoprecipitated proteins utilized in these ELISAs was 20 ng.
When screening the potential detection phage using the capture ELISA system, the phage was not biotinylated and therefore detected using the anti-M13 HRP antibody. Similarly, any reactivity with the commercial anti-TDP-43 antibody was recognized with the goat anti-rabbit IgG HRP secondary. Lastly, the quantity of immunoprecipitated proteins utilized in these ELISAs was 20 ng.
7
ELISA for Serum Antibody Quantification
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Polystyrene microtiter 96-well plates (Maxisorp; Nunc Nalgene) were coated with GFP (5 µg/ml in carbonate buffer, pH 9.6) and incubated overnight at 4°C. Plates were blocked with 3% BSA in PBS containing 0.05% Tween-20 (PBST). Samples were serially diluted 2-fold in blocking buffer in a range of 1∶200–1∶3,276,800, added to the wells, and incubated for 2 hours at 37°C. Plates were washed six times with PBST, and biotinilated goat anti-mouse IgG, IgM (Sigma), or monoclonal IgG1 or IgG2 (BD Pharmingen) were added to the wells (1 µg/ml) for 1 hour at 37°C. Avidin-HRP (1∶1000; Sigma) was then added and incubation continued for 30 min at 37°C. After six additional washes with PBST, 3,3′,5,5′ tetramethylbenzidine substrate (1-Step TMB; Pierce) was added, and the enzyme reaction proceeded for 20 min. The reaction was stopped with 2 M H2SO4. The absorbance was quantified in a microplate spectrophotometer at a wavelength of 450 nm. Serum antibody titers were determined by measuring the last dilution that resulted in three standard deviations above background.
8
Quantifying Chicken IL-7 Levels by ELISA
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The IL-7 levels were measured from the same serum samples which were used for the anti-EF-1α antibody analysis using a sandwich ELISA, as previously described (Panebra et al., 2021 ). Briefly, 96-well microtiter plates (Corning, MA) were coated with 5 μg/mL of purified chIL-7 monoclonal antibody (mAb) overnight at 4°C. The plate was washed twice and blocked with PBS containing 1.0% BSA for 1 h at room temperature. The chicken serums (12 chickens/group) diluted 1:10 in PBS containing 0.1% BSA were applied on a plate followed by 2 h of incubation at room temperature on the plate shaker. After 6 times washing, the plate was reacted with 1 μg/mL of biotin-labeled detecting chIL-7 mAb at 37°C for 1 h on the plate shaker. The plate was washed again, and the bounded antibodies were incubated with avidin-HRP in PBS containing 0.1% BSA (Sigma-Aldrich) (1:5,000) on a plate shaker at 37°C for 1 h followed by washing 6 times. All processes for plate washing, development, stop reaction, and OD measurement were performed via the same method as for indirect ELISA (Materials and Methods).
9
Electrochemical Exosome Detection
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For exosome detection, HCR-exosomes were prepared by adding approximately 1 million exosomes to 1 mL of a solution of HCR products and then shaken at 4 °C for 1 h (Fig. S2A in the Supporting Information). Next, 40 μL of HCR-exosome solution was added to the CD63-modified microelectrodes and incubated for 40 min at room temperature (Fig. S2B in the Supporting Information). Then, 2.5 μL of 10 μm/mL avidin−HRP (horseradish peroxidase coupled with avidin, Sigma Aldrich, MO, USA) was added to the microelectrodes. After 15 min at room temperature, the microelectrodes were rinsed to prepare for electrochemical analysis. An electrochemical analyzer (CHI 800B, TX, USA) was used for the analysis. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Neogen K-blue low-activity substrate, Shanghai, China) in H2O2 was used as the reaction solution38 (link). The electroreduction current was measured for 50 s with an applied voltage of 100 mV after the HRP redox reaction reached steady state.
10
MeHg-Induced UCH-L1 Quantification
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MeHg, avidin-HRP and avidin-agarose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Escherichia coli BL21 cells and trypsin were purchased from Promega Co. (Madison, WI, USA). Anti-ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). BPM was obtained from Dojindo (Kumamoto, Japan).
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