Levels of hFIX in plasma samples were determined by enzyme-linked immunosorbent assay (ELISA), and antibody concentrations specific to hFIX were measured by immunoglobulin subclass-specific immunocapture assay as published [12 (link)]. For ELISpot assay, splenocytes were harvested at 2 weeks after gene transfer and cultured. Splenocytes (300,000 per well) from individual mice were incubated in triplicate for 18 hours in an ELISpot plate coated with capture antibody for IFN-γ (R&D systems, Minneapolis, MN) with media alone, 10 µg/mL of recombinant hFIX or concanavalin A. A biotinylated secondary detection IFN-γ antibody (R&D systems, Minneapolis, MN) was added overnight at 4°C. Detection/Development utilized streptavidin-AP (R&D systems, Minneapolis, MN) in 100 µl of dilution buffer for 2 hours at room temperature, followed by color development with 100 µl of BCIP/NBT chromogen (R&D systems, Minneapolis, MN). IFN-γ producing cells were counted with the CTL-ImmunoSpotH S5 UV analyzer (Cellular Technology, Shaker Heights, OH).
Streptavidin ap
Manufactured by R&D Systems
Sourced in United States, United Kingdom
Streptavidin-AP is a conjugate of streptavidin and alkaline phosphatase. Streptavidin is a tetrameric protein that binds to biotin with high affinity. Alkaline phosphatase is an enzyme that catalyzes the dephosphorylation of various molecules.
Automatically generated - may contain errors
3 protocols using streptavidin ap
1
Quantification of hFIX Antibody Response
2
Quantifying Th1 Immune Memory with IFN-γ ELISpot
Check if the same lab product or an alternative is used in the 5 most similar protocols
ELISpot This assay is designed to determine the proportion of T cells that release IFN-γ after stimulation with SLA in order to quantify the level of stimulation of a specific Th1 polarity immune memory response. It was performed in a manner similar to that previously described [19 (link),27 (link)]. Heparinized blood samples were fractionated by centrifugation over lymphocyte separation medium. The PBMCs obtained were incubated at a density of 106 cells/mL for 3 days in multiscreen HTS filter plates (Millipore, Billerica, USA) previously coated with canine IFN-γ capture antibody (R&D System, Minneapolis, USA), in presence of 10 μg/mL ConA, or 10 μg/mL SLA antigens, or with medium alone, in a humidified 37 °C CO2 incubator. The quantity of IFN-γ was revealed with a specific biotinylated antibody and incubation with Streptavidin-AP and the BCIP/NBT Chromogen (R&D System, Minneapolis, USA). The number of specific spots was determined by an automated ELISpot reader. ConA was used as a positive control and the medium alone was used as a negative control. The data presented are the number of spots per 2 × 105 cells after stimulation with SLA minus the equivalent value obtained with the negative control using medium alone.
+ Expand
3
ELISpot Assay for Measuring Cytokine Secretion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ELISpot plates (Millipore, Watford, UK) were coated overnight with IL-2 capture antibody (R&D Systems, Abingdon, UK), washed, and incubated overnight in blocking buffer (1% BSA in PBS). Cell density was adjusted to 4-6 x 10 6 PBMC/mL, and 100 μL of cells incubated with full length rHuPH20, A33 (positive control), or buffer for 8 days. ELISpot plates were washed, incubated with biotinylated anti-mouse detection antibody (R&D Systems, Abingdon, UK) for 1.5 h at 37°C, followed by incubation with streptavidin-AP (R&D Systems, Abingdon, UK) for 30 min at room temperature. Plates were then incubated with BCIP/NBT substrate (R&D Systems, Abingdon, UK) for 30 min at room temperature. The wells were washed with distilled H 2 0, dried, and scanned on an Immunoscan ® Analyser (Cellular Technology Limited, Cleveland, OH, USA) and spots per well were determined using Immunoscan ® software, Version 5.
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!