Hp 6017

Ninety-six-well plates (Costar 3590) were coated with mouse anti-human IgG (against C_H3 domain) (MCA878G, 1 μg/ml, AbD serotec, Oxford, UK) or NIP-BSA. Plates were blocked with 0.1% BSA in PBS, and supernatants from HEK293E cells were transiently transfected with 1 μg of vaccine plasmids, or purified vaccine proteins were added to wells in triplicates. Plates were now incubated with biotinylated mAb against IgG (Fc fragment) (HP-6017, 1 μg/ml, Sigma-Aldrich, Germany) or a C179 mAb specific for stem HA (1 μg/ml, kind gift from Yoshinobu Okuno, Osaka University, Japan) followed by biotinylated anti mouse IgG2a [1 μg/ml IgG2a(a) 553502, BD Pharmingen]. Next, plates were incubated with streptavidin alkaline phosphatase (1:3000, GE Healthcare, USA), developed with phosphatase substrate (P4744-10G, Sigma-Aldrich, Germany), and read at 405 nm with a Tecan reader using the Magellan v5.03 program.

HEK293E cells were transfected with plasmids encoding dimeric fusion proteins that expressed a scFv against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx)^{63 (link)} linked to the human Ig-hinge regions h1 and h3, and CH3 from human IgG3 as dimerization unit^{27 (link)}, and to the HA stem derived from either H1 or H7 as antigenic unit^{64 (link)} to form scFv^αphOx-CH3-HA stem proteins. phOx (E0753-1G, Sigma) was coupled to BSA. The resulting phOx-BSA (1 µg/ml) was used as coat, and supernatant with scFv^αphOx-C_H3-HA stem proteins was added and incubated for 2 hours. The scFv^αphOx -C_H3-HA stem supernatant was exchanged with fresh supernatant twice to obtain a saturated coating with protein dimers. The presence of scFv^αphOx -C_H3-HA stem in the ELISA was confirmed through binding of mAb towards the human IgG3-based dimerization motif (anti-human IgG-Biotin, 1 µg/ml, clone HP-6017, B3773, Sigma) and rabbit anti-HA stem mAb (1:3000) detected with anti-rabbit IgG Ab-ALP. Mice sera were serially diluted 3-fold in ELISA buffer, starting at 3:50. HA stem-specific antibodies were detected with anti-mouse IgG-ALP conjugate.