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2 protocols using chicken egg ova elisa kit

1

Quantifying Antigenic OVA Release

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In vitro release samples collected and analyzed by SE-HPLC were further analyzed by enzyme-linked immunosorbent assay (ELISA) to quantify the amount of antigenic OVA in each sample. Samples were collected at time points of 1, 3, 5, 7, 14, and 21 days. Antigenic OVA was detected using a commercial chicken egg OVA ELISA kit (Alpha Diagnostic International, USA), which was used according to the manufacturer’s instructions. Samples were diluted with the sample diluent provided with the kit to fall within the working range of the assay and then assayed in duplicate. The dilution was based upon the calculated concentration of OVA in the samples determined by SE-HPLC. The ELISA plate was read at 405 nm using a Synergy Neo plate reader with Gen5 software (Biotek Instruments Inc., USA).
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2

Multiparametric Flow Cytometry Analysis

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Fluorophore directly conjugated anti-CD45.1, anti-CD45.2, anti-CD4, anti-CD8, anti-CD25, anti-Foxp3, and anti-B220 monoclonal antibodies (mAbs) were purchased from eBioscience (San Diego, CA, USA). Anti-mouse CD41 mAb directly conjugated with phycoerythrin (PE) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse CD42b mAb conjugated with DyLight 649 was purchased from Emfret Analytics (Eibelstadt, Germany). A Foxp3 transcription factor staining buffer set was purchased from eBioscience. Mouse BD Fc Block (purified rat anti-mouse CD16/CD32) was purchased from BD Pharmingen (Franklin Lakes, NJ, USA). The EasySep mouse SCA1 positive selection kit was purchased from STEMCELL Technologies (Cambridge, MA, USA). X-VIVO 10 media were purchased from Lonza (Walkersville, MD, USA). The QIAamp DNA blood mini kit was purchased from QIAGEN (Germantown, MD, USA). GoTaq green master mix was purchased from Promega (Madison, WI, USA). OVA protein was purchased from Sigma (St. Louis, MO, USA; catalog no. A2512, grade VI). 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; a zwitterionic detergent) was purchased from MP Biomedicals (Solon, OH, USA). A chicken egg OVA ELISA kit was purchased from Alpha Diagnostic International (San Antonio, TX, USA). Recombinant human full-length FVIII (rhfF8; Kogenate FS) was from Bayer Healthcare (Whippany, NJ, USA).
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