Mouse tissues were used in this study to avoid epitope degradation that may occur when tissues are not immediately processed post mortem. Mouse brain and kidney lysates were made as described elsewhere [51 ] and the protocol was adapted from this reference. Protocol details are provided in the supplemental methods . In brief, plates were coated with 10 μg/mL of lysateor 1 μg/mL of purified antigen in bicarbonate buffer overnight, then blocked. Dilutions of rhAbs or patient plasma were added overnight, followed by incubation with biotinylated anti-human IgG (eBioscience, San Diego, CA) then streptavidin-HRP (BD Pharmigen, San Jose, CA) and detection by TMB substrate according to manufacturer recommendations (eBioscience, San Diego, CA). Background signal (typically 0.05 to 0.09, defined as wells without primary antibody) was subtracted from each measured well. Patient rhAbs were defined as positive by ELISA if the average absorbance of the rhAb at 20 and 10 μg/mL was more than two standard deviations higher than the average absorbance of the healthy donor rhAbs at 20 μg/mL.
Streptavidin hrp
Manufactured by BD
Sourced in United States
Streptavidin-HRP is a conjugate of streptavidin, a protein that binds to biotin, and horseradish peroxidase (HRP), an enzyme that can catalyze colorimetric or chemiluminescent reactions. This conjugate is commonly used in various bioanalytical techniques, such as enzyme-linked immunosorbent assays (ELISAs), Western blotting, and immunohistochemistry, to detect and quantify biomolecules that are labeled with biotin.
Automatically generated - may contain errors
Lab products found in correlation
Aec substrate, by BD (8 mentions)
Elispot plate, by Merck Group (6 mentions)
Imark microplate reader, by Bio-Rad (5 mentions)
R35 118, by BD (4 mentions)
Biotin rat anti mouse igg1, by Thermo Fisher Scientific (4 mentions)
Duoset elisa kit, by R&D Systems (4 mentions)
Phytohaemagglutinin, by Merck Group (4 mentions)
Immobilon p plates, by Merck Group (4 mentions)
Tmb substrate set, by BD (3 mentions)
Biotinylated anti mouse ifn γ, by BD (3 mentions)
Nunc maxisorp 96 well plate, by Thermo Fisher Scientific (3 mentions)
Maxisorp plate, by Thermo Fisher Scientific (3 mentions)
Biotinylated secondary antibody, by Vector Laboratories (3 mentions)
Histopaque 1077, by Merck Group (3 mentions)
Eotaxin, by R&D Systems (2 mentions)
T per buffer, by Thermo Fisher Scientific (2 mentions)
Biotinylated rat anti igg1 clone a85 1, by BD (2 mentions)
Rat anti ige clone r35 118, by BD (2 mentions)
Supersensitive tmb, by Merck Group (2 mentions)
Rantes, by R&D Systems (2 mentions)
171 protocols using streptavidin hrp
1
ELISA Assay for Autoantibody Detection
2
Quantification of Allergic Immune Markers
3
Quantification of Allergic Immune Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytokine, chemokine and antibody concentrations were measured via ELISA. Plasma collected at the time of sacrifice was assayed for OVA-specific IgE and IgG1 similarly to previously described.30 (link) Briefly, plates were coated with 100 μg/ml OVA, and blocked with 1% BSA, prior to incubation with diluted plasma. Bound antibody was detected with biotinylated rat anti-IgG1 (clone A85–1, BD Pharmigen) or rat anti-IgE (clone R35–118, BD Pharmigen), followed by addition of streptavidin-HRP (BD Pharmigen) and supersensitive TMB (Sigma-Aldrich) as a substrate. Lung homogentates were prepared from half of the left lung as previously described27 (link) in T-PER buffer (ThermoFisher Scientific, Waltham, MA, USA). ELISAs for IL-5 (eBioscience), eotaxin (R&D Systems, Minneapolis, MN, USA), and RANTES (R&D Systems) were performed according to the manufacturers’ instructions.
4
Inhibition ELISA for Cross-Reactivity Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Inhibition ELISA showed that pre-incubation of sera with Fel d 1 or hamster extract did not decrease IgE binding to Can f 1, indicating no allergenic cross-reactivity (Figure 3). by five washes with washing solution (0.05% Tween-20 in PBS, pH 7.0). The samples were incubated for 2 h at room temperature in blocking solution (10% fetal bovine serum in PBS) to inhibit unspecific interactions and then washed five times. Subsequently, 100 µL of patient serum diluted 1:10 in blocking solution was added to each well, and the plate was incubated for 16 h at 4°C. After five washes, biotinylated human anti-IgE (Vector Laboratories, Burlingame, CA, USA) was diluted 1:1,000 in blocking solution and then it was diluted 1:250 with streptavidin-HRP (BD PharMigen, San Jose, CA, USA). The reaction was carried out for 1 h at room temperature, followed by seven washes. Then, 3,3′,5,5′-tetramethylbenzidine (BD PharMigen, San Jose, CA, USA) was added for 10 min in a dark room, after which 100 µL 2 N H 2 SO 4 was added to stop the reaction. Optical density (OD) was measured at 450 nm, and the value was multiplied by 1,000 to derive the log value from which the relative concentrations of specific IgE were derived. The cut-off value for a positive result was set as mean + 2 standard deviation (2SD) of the negative control log(OD × 1,000).
5
Quantification of Chemerin Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemerin levels in conditioned media or in epidermis lysates was quantified by human or mouse-specific ELISA. High-binding ELISA strips (Nunc) were coated with mouse-anti-human chemerin mAbs (MAB23241), or goat-anti-mouse Abs (AF2325) (both from R&D Systems) in Tris-buffered saline (50 mM Tris-HCl pH 9.5, 150 mM NaCl). The plates were then washed with PBS containing 0.1% Tween 20, and nonspecific protein-binding sites were blocked with 3% BSA in PBS. Human or mouse recombinant chemerin was used as a standard. Chemerin was detected using biotin-conjugated goat anti-human chemerin Abs (BAF2324) or biotin-conjugated rat-anti mouse chemerin mAbs (BAM2325) followed by streptavidin-HRP (BD Science). The reaction was developed with TMB substrate (BD Science).The ELISA detects both the 163S and 157S chemerin. Alternatively, chemerin in mouse skin homogenates (100%) and plasma samples (diluted 1/200) was detected by comercially available ELISA (R&D Systems), according to the manufacturer’s instructions. The levels of chemerin in plasma or skin homogenates and epidermis extracts were undetectable in chemerin KO mice.
6
Antigen-specific Antibody and Cytokine ELISAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TM E/S antigen-specific serum IgG1 and IgG2c ELISAs were carried out as described [15 (link)]. Briefly, serum was added to ELISA plates that had been coated overnight with TM E/S antigen (5ug/ml), washed and blocked in 5% fetal calf serum. Following a 2h 37°C incubation, plates were sequentially washed, incubated for 1 hour at room temperature with secondary antibodies (BD Biosciences: biotinylated anti-mouse IgG1, clone A85-1 or biotinylated anti-mouse IgG2c, clone R19-15), washed and incubated with streptavidin HRP (BD Pharmingen) for 30 minutes at room temperature and finally detected with TMB substrate (eBioscience). Enzymatic reactions were stopped with 1N H2SO4 and OD450 measured on a Spectramax (Molecular Devices) spectrophotometer.
IgE ELISA was carried out by using the IgE ELISA kit from Thermofisher according to manufacturer’s instructions. OD values were measured using a Spectrophotometer (Versamax).
IFNγ ELISA was carried out with the Mouse IFN gamma ELISA Ready-SET-Go! Kit (eBioscience, cat #88–7314) according to manufacturer’s instructions. TSLP (Mouse TSLP Quantikine ELISA kit, R&D systems) and IL13, IL10 and IL4 were measured using a Milliplex Map kit (Millipore) according to manufacturer’s instructions.
IgE ELISA was carried out by using the IgE ELISA kit from Thermofisher according to manufacturer’s instructions. OD values were measured using a Spectrophotometer (Versamax).
IFNγ ELISA was carried out with the Mouse IFN gamma ELISA Ready-SET-Go! Kit (eBioscience, cat #88–7314) according to manufacturer’s instructions. TSLP (Mouse TSLP Quantikine ELISA kit, R&D systems) and IL13, IL10 and IL4 were measured using a Milliplex Map kit (Millipore) according to manufacturer’s instructions.
7
Serum Antibody Detection by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serially diluted serum collected from the indicated mice were transferred to 96-well ELISA plates Nunc MaxiSorp™ (Thermo Fisher Scientific, Waltham, MA) precoated with rMOGm or purified anti-IgG1 antibodies. After extensive washing, bound Ig was detected by a sandwich consisting of a biotinylated allotype- and isotype-specific anti-mouse IgG1 labelled with streptavidin-HRP (BD Biosciences, San Jose, CA). A colorimetric reaction was performed with an ATBS (2,2’-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate and absorbance was measured at 405 nm.
8
IFNγ ELISpot Assay for Tumor-Specific T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ELISpot plate (BD Biosciences) was treated with sterile-filtered 70% ethanol before being washed three times with sterile PBS 1×. IFNγ capture antibody (BD Biosciences no. 551881) was plated and the plate was sealed and left to incubate at 4°C overnight. Positive control wells were also plated with anti-CD3ε (BioLegend no. 100340). The plate was washed three times with sterile PBS and blocked with 10% (v/v) FBS in PBS overnight at 4°C. IFNγ-stimulated 6694c2 or B16F10 cells were plated, except in the unstimulated and positive control wells. Pancreatic draining lymph nodes were excised from treated mice bearing orthotopic tumours, and lymph nodes were macerated to form a single-cell suspension. Lymph node cells (one-third of lymph node per well) were plated on top of pre-plated tumour cells with human IL-2 (PeproTech), and positive control wells also received anti-CD28 (BioLegend no. 102116). Plate was incubated at 37°C for 24 h before being washed with sterile water followed by PBST. IFNγ detection antibody (BD Biosciences no. 551881) was added, and plate was incubated for 2 h at room temperature. Wells were washed with PBST, and streptavidin-HRP (BD Biosciences) was added. Plate was incubated for 1 h at room temperature before being washed with PBST and PBS. AEC chromagen substrate (BD Biosciences) was added, and plate was developed. Plate was dried and analysed.
9
Cytokine and Antibody Measurements
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytokine concentrations were measured in cell culture supernatants using either FlowCytomix (eBioscience) or a LegendPlex Mouse Th1/Th2 Panel (BioLegend) flow cytometry multi-analyte detection system for IL-4, IL-2, IL-5, and IL-13 per the manufacturer’s instructions. Serum IgE (and purified mouse IgE standard; BD) was captured overnight on a plate coated with 2 µg/ml rat anti–mouse IgE (R35-72; BD) and detected with biotin rat anti–mouse IgE at 1 µg/ml (R35-118; BD), streptavidin HRP (BD), and ABTS One Component HRP Microwell substrate (SurModics). H. polygyrus antigen (HEX) was obtained by homogenizing adult worms in PBS. Serum antigen-specific IgG1 was captured on a plate coated with 5 µg/ml HEX and detected using biotin rat anti–mouse IgG1 (Invitrogen), streptavidin HRP, and ABTS.
10
Western Blot Analysis of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purity of the proteins and the efficiency of in vivo biotinylation were analyzed by western blotting. 30 μL of each purified protein and 6 μL of 5× reducing SDS loading buffer were added to 1.5-mL tubes and denatured at 96°C for 5 min. The samples were run on SDS-PAGE gel in SDS running buffer at 100 V for 2 hr. The gel was then used for western blotting at 100 V for 1 hr 30 min. The membrane was blocked with 1% BSA and hybridized with a specific horseradish peroxidase (HRP). Anti-6× His-tag antibody HRP was used to detect the protein construct, and streptavidin-HRP (BD Biosciences, San Jose, CA) was used to determine the success of the in vivo biotinylation of the protein construct. Secondary hybridization was performed with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA), an enhanced chemiluminescent (ECL) substrate for the HRP enzyme.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!