Control mAbs of known binding epitope [5 (link), 15 (link), 16 (link)] were biotinylated using NHS-PEG4-Biotin, No-Weigh™ Format kit (Thermo, 21329) at 3 mg/ml in DPBS. Biotinylated mAbs (B-mAbs) were normalized to 15 μg/ml in DPBS, and 1:2 serial dilutions were performed in ELISA Buffer in a separate plate (BD Falcon, 353077) before being added to the reaction plate, prepared using monobulk antigen as described above. After blocking, one volume of ELISA Buffer was added to the reaction plate followed by one volume of B-mAb dilution. Plates were incubated lh with shaking at RT, then washed three times. HRP-conjugated Avidin D (Vector Labs, A-2004) was diluted 1:10,000 in ELISA Buffer and loaded onto plates. Plates were then processed as described above. Concentrations of respective B-mAbs at which elicited 450 nm absorbances were 2.0–3.0 (nominally, “EC75”) were selected for use in subsequent assays described below.
Hrp conjugated avidin d
Manufactured by Vector Laboratories
Sourced in United States
HRP-conjugated avidin D is a reagent that binds to biotin-labeled molecules. It can be used in various detection and labeling applications, such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti tnf α, by BioLegend (3 mentions)
Anti il 6, by BioLegend (3 mentions)
Biotinylated detection mabs, by BioLegend (3 mentions)
Mouse il 10 duoset elisa kit, by R&D Systems (2 mentions)
Anti il 1β, by BioLegend (2 mentions)
Mayer s hematoxylin solution, by Merck Group (1 mentions)
Aec kit, by Vector Laboratories (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
7 protocols using hrp conjugated avidin d
1
Biotinylated mAbs Binding Analysis
2
ELISPOT Assay for LCMV-Specific IgG Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ELISPOT assay was performed as previously described [17 (link)]. Briefly, filter plates were coated overnight at RT with LCMV-infected BHK-cell lysates. Plates were washed with PBS-T and PBS and blocked with RPMI with supplements for 2 h at RT (10% FCS). Splenocytes and bone marrow cells were serial diluted onto filter plates and incubated for 8 h 37°C in humidified 5% CO2 incubator. After washing with PBS and PBS-T the plates were incubated with biotinylated anti-IgG (Vector Laboratories, Burlingame, CA, USA) overnight at 4°C. The plates were then incubated with HRP-conjugated avidin-D (Vector Laboratories, Burlingame, CA, USA) for 60 min at RT. Following washing with PBS-T and PBS plates were stained with AEC working solution and rinsed with dH2O.
3
Localization of Human Norovirus Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections from the gastrointestinal tracts of six dogs were deparaffinized through baths of LMR-SOL (1-bromopropane, 2-methylpropane-2-ol, and acetonitrile), followed by rehydration with successive baths of 100, 90, 70, and 50% ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in PBS. Nonspecific binding was blocked with 3% bovine serum albumin (BSA) in PBS. H and A antigen detection was then performed as previously reported (23 (link)). To assess the ability of VLPs to bind to tissue sections, after blocking, 1 μg/ml VLPs was incubated with the sections overnight at room temperature. Anti-HuNoV primary antibody was then incubated with the tissue sections for 1 h at 37°C. After three washes in PBS, sections were incubated with secondary anti-rabbit biotinylated antibody (Vector Laboratories, Burlingame, CA) diluted in 1% BSA in PBS for 1 h. Sections were washed three times in PBS prior to addition of HRP-conjugated avidin D (Vector Laboratories, Burlingame, CA) also diluted in 1% BSA in PBS. Substrate was added to the slides (AEC kit; Vector Laboratories, Burlingame, CA), followed by Mayer's hematoxylin solution (Merck, Whitehouse Station, NJ) for contrast staining.
4
Cytokine Detection via ELISA
5
Quantification of Cytokine Levels in Cell Supernatants
6
Quantitative IL-21 ELISA Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Maxisorp 96-well plates were precoated overnight at 4°C with 2 μg/mL purified anti–human IL-21 capture mAb (clone J148-1134; BD Biosciences) in 100 μL bicarbonate buffer pH 9.6 per well. The next morning, the unbound antibody was removed, and the coated plates were blocked for 2 h with 300 μL per well of PBS with 2% bovine serum albumin at 37°C. The plates were then washed four times with PBS supplemented with 0.05% Tween 20, added serial 2-fold dilutions of test plasma samples in duplicates and a dilution series of a IL-21–Fc standard, and incubated for 2 h at RT. Plates were washed and added 100 μL of anti–IL-21–biotin detection mAb (clone I76-539; BD Biosciences) at a 1:2,000 dilution and incubated for 2 h at RT. After washing, the plates were added HRP-conjugated Avidin D (Vector Laboratories, Burlingame, CA, USA) at a 1:2,000 dilution followed by TMB substrate (KPL) in sequential steps. The reaction was stopped by the addition of 20 μL of 1 M H2SO4, and the absorbance read at 450 nm using a Bio-Tek Synergy HT multimode microplate reader. Baseline plasma samples for each test subject collected prior to IL-21 administration were included to determine background values. The lower detection limit of IL-21–Fc was 15.6 pg/mL.
7
Cytokine Detection in Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the detection of cytokines in the cell culture supernatants or sera, ELISAs were performed as described previously (17) , using the primary capture mAbs anti-TNFα, anti-IL-6, and anti-IL-1β and their corresponding biotinylated detection mAbs (Biolegend, San Diego, CA). Other ELISA reagents included: recombinant mouse TNFα, IL-6, and IL-1β as standards (Biolegend, San Diego, CA), HRP-conjugated avidin D (Vector Laboratories, Burlingame, CA), and TMB microwell peroxidase substrate and stop solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD). IL-10 cytokine was measured using Mouse IL-10 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. Biological replicates were used, representing independent BMDM cultures from individual mice.
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!