Ap conjugated donkey anti mouse igg h l minimal cross

Direct ELISA was performed against the SARS-CoV-2 spike [30 (link)]. Maxisorp 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight with 1 μg/mL of spike in NaHCO₃ buffer (50 mM, pH 9.6), washed, and blocked with PBST (Phosphate buffer saline, 2% BSA, 0.05% Tween 20) at room temperature for 1 h. The secondary antibodies: AP-conjugated Donkey anti-human IgG (Jackson ImmunoResearch, Baltimore, PA, USA, Cat# 709-055-149, lot 130049) or AP-conjugated Donkey anti-mouse IgG (H + L) minimal cross (Jackson ImmunoResearch, USA, Cat# 715-055-150, lot 142717) were applied, followed by the addition of PNPP substrate (Cat No. N1891, Sigma, Rehovot, Israel).

Direct ELISA was performed against the SARS-CoV-2 S1 subunit (expressed and purified as recently described^{11 (link)}) for binding evaluation and for Ab concentration determination. Maxisorp 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight with 1 μg/ml of S1 protein in NaHCO₃ buffer (50 mM, pH 9.6), washed, and blocked with PBST at room temperature for 1 h. Human Abs were visualized by AP-conjugated Donkey anti-human IgG (Jackson ImmunoResearch, USA, Cat# 709-055-149, lot 130049) used at 1:1000 and further developed with PNPP substrate (Sigma, Israel, Cat# N1891). For the quantification of MD65 Ab presence in plasma or sera samples, standard curve included purified MD65 IgG-YTE Ab at the concentration range of 2–3000 ng/ml (diluted in PBST). Correct ELISA-based quantification was confirmed by four non-immune sera samples (collected prior to Ab-administration) containing exogenously added known concentration of the Ab. Standard curves confirmed as above were calculated for each individual ELISA plate/data set.
The endogenous humoral response was tested in mice sera samples collected at 14 dpi, evaluated by ELISA against SARS-CoV-2 spike, S1 and RBD essentially as described above, using AP-conjugated Donkey anti-mouse IgG (H + L) minimal cross (Jackson ImmunoResearch, USA, Cat# 715-055-150, lot 142717) used at 1:2000.