His tag igg

To measure antibody responses competing with the human IgG1 CR9114 monoclonal antibody binding to a conserved epitope in the stem region of influenza HA [11 (link)], a modified ELISA protocol was used. Maxisorp 96-well plates (NUNC) were coated with purified polyclonal rabbit anti His-Tag IgG (Genscript USA Inc., NJ, US) O/N at 4°C followed by washing. After blocking with PBS/BSA and washing, plates were incubated with a titrated amount of His-Tagged HA of A/Brisbane/59/07 (produced in HEK293F cells) for 2 hours at RT. Plates were washed and individual serum samples were added to the plate in duplicate, serially diluted in PBS/BSA and incubated for 1 hour at RT, followed by addition of a titrated amount of biotinylated human CR9114 and incubation for another hour at RT. After washing, streptavidin-HRP was added and incubated for 1 hour at RT, followed by washing and OPD development. The CR9114 competition of each sample was quantified as the slope of the linear regression of OD value on the log10 dilution for the duplicate series. Positive and negative controls consisted of competition with monoclonal antibodies CR9114 (at 2.5μg/ml) [11 (link)].

To quantify CR9114 epitope-competing antibodies 96-well Maxisorp plates (Nunc^™, Thermo Scientific) were coated o/n at 4 °C with purified polyclonal rabbit anti His-Tag IgG (GenScript USA Inc. Cat. No. A00174–200). Plates were washed using an ELx405 automated plate washer (BioTek) programmed for 3 washes with 300 μl of PBS supplemented with 0.05% Tween-20 (Calbiochem, Merck Millipore, Cat. No. 655204) and subsequently blocked with 2% BSA in PBS for 1 hour at RT. After washing, the plates were incubated for 2 hours with His-tagged FL HA of A/Brisbane/59/2007 (in-house production), washed again, and serum was added in duplicate followed by a 2-fold serial dilution in blockbuffer. After the first hour of incubation at RT, the competing biotinylated human IgG1 CR9114 was added (0.02μg/ml). The plates were incubated for 1 additional hour at RT and washed again before adding streptavidin-HRP for 1 hour. The plates were washed, developed using OPD substrate (Thermo Scientific, Tablets: Cat. No. 34006 Buffer Cat. No. 34062) and stopped after 10 minutes with 1M H₂SO₄. OD was measured at 492nm by a Powerwave HT plate reader (BioTek) and fitted using a 4-parameter logistic curve. The CR9114 competition of each sample was quantified as the slope of the linear regression of OD value on the log10 dilution for the duplicate series.

To determine stem-binding antibody responses, we measured CR9114-competing antibody responses as described before (44 (link)). Briefly, Maxisorp 96-well plates (Merck) were coated with purified polyclonal rabbit anti His-Tag IgG (Genscript, NJ, USA) O/N at 4°C followed by washing. After blocking and washing, plates were incubated with a titrated amount of His-Tagged HA of A/California/07/09 (in house produced and purified) for 2 h at RT. Plates were washed, and plasma was added to the plate in duplicate, serially diluted in block buffer, and incubated for 1 h at RT, followed by addition of a titrated amount of biotinylated human IgG1 CR9114 (produced and purified in house) and incubation for another hour at RT. After washing, streptavidin–HRP was added and incubated for 1 h at RT, followed by washing and OPD (Thermo Scientific, Bremen, Germany) development. The colorimetric reaction was stopped after 10 min by adding 1M H₂SO₄. The optical density (OD) was measured at 492 nm, and standard curves were created using a four-parameter logistic curve. The CR9114 competition of each sample was quantified as the slope of the linear regression of OD value on the log 10 dilution for the duplicate series.