Bvd6 24g2

Culture supernatant cytokines (mouse IL-4, IL-5 and IFN-γ) were measured by two-site sandwich ELISA. The Abs for coating the plates and the biotinylated secondary mAbs were as follows: for IL-4, rat mAb anti-mouse IL-4 (BVD4-1D11, BD Biosciences, San Diego, CA) and biotinylated mAb anti-mouse IL-4 (BVD6-24G2, BD Biosciences); for IL-5, rat mAb anti-mouse/human IL-5 (TRFK5, BD Biosciences) and biotinylated mAb anti-mouse IL-5 (TRFK4, BD Biosciences); for IFN-γ, polyclonal rabbit anti-mouse IFN-γ Ab (prepared in our laboratory) and biotinylated rat mAb anti-mouse IFN-γ (XMG1.2, BD Pharmingen). HRPO-conjugated Streptavidin was purchased from Zymed (San Francisco, CA). Levels of human TNF-α were determined by an ELISA system that was developed in our laboratory. Levels of human IL-10 were determined with human IL-10 ELISA Ready-SET-Go kit (Thermo Fisher Scientific, Waltham, MA). Eotaxin and thymus and activation-regulated chemokine (TARC) were measured with human CCL11/Eotaxin DuoSet ELISA Development Systems (R&D Systems, DY320) and human CCL17/TARC DuoSet ELISA Development Systems (R&D Systems, DY364), respectively.

T cell memory responses from immunized splenocytes were quantified using enzyme-linked immunospot (ELISpot) assay. Multiscreen-IP filter plates (MSIPS4510, Merck) were coated with anti-mouse IFNγ antibody (5 μg/mL, 100 μL/well, clone AN18, MABTech, Nacka Strand, Sweden) or anti-mouse IL4 antibody (5 μg/mL, 100 μL/well, BVD4-1D11, BD Biosciences) overnight at 4 °C, washed 5 times with 200 μL/well sterile PBS and blocked with complete RPMI (150 μL/well) for a minimum of 1 h at 37 °C. Then, 15 μg/mL of MSP4/5 was used for recall with 5 × 10⁵ splenocytes. Medium alone was used as the negative control and 1 μg/mL Concavalin A as a positive control. IFNγ was labelled with biotin anti-mouse IFNγ antibody (1 μg/mL, 100 μL/well, R4-6A2-Biotin, MABTech) or biotin anti-mouse IL4 (1 μg/mL, 100 μL/well, BVD6-24G2, BD Biosciences) for 2 h at room temperature. This was then labelled with streptavidin-alkaline phosphatase (ALP) (1:1000, 100 μL/well, 3310-10, MABTech) or ExtrAvidin-ALP (1:3000, 100 μL/well, Sigma-Aldrich) for 1.5 h at room temperature. Spots were developed with an ALP conjugate substrate kit (Bio-Rad, Hercules, CA, USA) and imaged and counted with an ELISpot reader and software (AID, Strasburg, Germany).