ELISA Assay for Trimeric Env Glycoprotein

Titer of IgG antibodies binding to trimeric Env glycoprotein was determined by ELISA using a D7324-tagged version of the recently described soluble HIV-1 Env trimer BG505 SOSIP.664 gp140 and following the methods described by Sanders et al [22 (link)]. Briefly, Maxisorb Elisa plates (Nunc) were coating with D7324 antibody (10 μg/mL) and blocked using TBS+10%FBS for two hours at room temperature. After washing, the BG505 SOSIP.664 protein was added at 1ng/ml in blocking buffer and incubated overnight at 4°C. Then, plates were washed and blocked again using TBS/2% skimmed milk. After washing, plasma samples diluted 1/1000 in TBS/5%FBS/2%skimmed milk were added and incubated for two hours at room temperature. HRP-Goat anti-human IgG (Fc specific) (Jackson-Immunoresearch) was used as secondary antibody. The reaction was revealed using 3, 3′,5, 5′-Tetramethylbenzidine (Sigma-Aldrich) and stopped using 2M of H₂SO₄. 2G12 antibody was used as standard and the results are showed as arbitrary units. The IgGb12 antibody, which does not bind to the BG505 SOSIP.664, was used as negative control. Plasma samples were assayed in parallel in D7324 antibody coated and antigen free wells to evaluate background. The signal obtained in these wells was subtracted to the signal obtained with antigen.