Mouse anti human igg1

RBD-specific IgG antibodies were determined using Enzyme-Linked Immunosorbent Assay (ELISA) as previously described (6 (link), 15 (link), 16 (link)). To determine IgG subclasses antibodies (IgG1, IgG2 IgG3 and IgG4), 100 µL of serum samples were added to the plate which were initially diluted to 1:10 and then 3-fold, 8 serial dilution was performed. Plates were subsequently incubated at 37°C for an hour. Mouse anti-human IgG1 (1:3000), IgG2 (1:4000), IgG3 (1:3000) and IgG4 (1:3000) Fc specific HRP (Southern Biotech) were added to the plates and incubated for one hour at room temperature. Subsequently, Ortho- phenylenediamine in 0.1 M sodium citrate buffer (100 µL; pH 4.5) and 30% hydrogen peroxide were added to the plate. The reactions were stopped after 20 minutes by adding 1 M H₂SO₄ (25 µL) and endpoint titers were determined as the reciprocal interpolated dilutions of the samples at 492 nm that were 0.4 above the background.

Mouse anti-human IgG₁ (#9054-01), mouse anti-human IgG₂ (#31-7-4), mouse anti-human IgG₄ (#9200-01), and mouse anti-human IgE (#9250-01) were obtained from Southern Biotechnology (Birmingham, AL). Mouse anti-human IgG₃ (#05-3600) was obtained from Invitrogen (Carlsbad, CA) and mouse anti-human IgM (#555856) and mouse anti-human IgA (#555886) were obtained from BD Biosciences (San Jose, CA). Secondary detection reagents including biotin-mouse anti-human kappa light chain (#555790) and biotin-mouse anti-human lambda light chain (#555794) were obtained from BD Biosciences (San Jose, CA). R-Phycoerythrin-conjugated streptavidin (#016-110-084) and R-Phycoerythrin-conjugated donkey F(ab’)₂ anti-mouse IgG (H + L) (#715–116-150) were obtained from Jackson ImmunoResearch (West Grove, PA).

CHK-152 was diluted in a sodium bicarbonate buffer (pH 9.3) and adsorbed overnight at 4°C on a Maxisorp immunocapture ELISA plate. Wells were washed with PBS with 0.05% Tween 20 and incubated with blocking buffer for 1 h at 37°C. CHIKV 181-25 was captured for 1 h at room temperature. Hyperimmune IgG samples were serially diluted in blocking buffer, added to wells, and incubated for 1 h at room temperature. Plates were washed and then incubated with biotin-conjugated mouse anti-human IgG1 (Southern Biotech clone 4E3), mouse anti-human IgG2 (Southern Biotech clone HP6002), mouse anti-human IgG3 (Southern Biotech clone HP6050), mouse anti-human IgG4 (Southern Biotech clone HP6023), or goat anti-human IgG Fc-multiple species cross-adsorbed (Southern Biotech) antibody for 1 h at room temperature. Wells were washed and incubated with HRP-conjugated streptavidin at room temperature for 1 h. Following washing, the assay was developed as described for above. The endpoint dilution was determined as described above.