Goat anti mouse igm hrp

ELISAs were performed in microplates (655061; Fisher Scientific, Carlsbad, CA, USA) coated overnight at 4 °C with 2 µg/mL (50 µL/well) of S1, N, and RBD proteins diluted in PBS 1X pH 7.4. For variants, microplates were coated overnight at 4 °C with 2 µg RBD proteins based in Wuhan isolate, Alpha variant, and Beta variant diluted in PBS 1X pH 7.4. RBD expressed in Expi293 cells was used as a control.
Plates were washed 3 times with PBS/0.1% Tween 20 and then blocked with PBS containing 5% (w/v) skim milk for 1 h. Next, serum diluted 1:100 in PBS containing 5% (w/v) skim milk was added, and plates were incubated for 2 h. After washing, the secondary antibodies goat anti-mouse IgM-HRP (G21040, Invitrogen) and goat anti-mouse IgG-HRP (62-6820, Invitrogen) were added at a dilution of 1:5000. For isotype determination, the secondary antibodies rabbit anti-mouse IgG1-HRP (61-0120, Invitrogen) and rabbit anti-mouse IgG2a-HRP (61-0220, Invitrogen) were added at a dilution of 1:2000. Plates were incubated for 1 h at 37 °C. After washing five times, bound conjugates were visualized using o-phenylenediamine (P9029; Sigma-Aldrich, St. Louis, MO, USA) and H₂O₂ (Sigma) as substrates, allowing the reaction to proceed for 15 min. The reaction was stopped with 50 μL of 2 N H₂SO₄ Optical density was measured at 450 nm using an ELISA Lektor (Multiskan FC, Thermo Scientific, Whaltan, USA)

Total proteins were extracted from tissues using TX-100 buffer (1% Triton X-100, 50mM Tris pH8.0, 150mM NaCl, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche, Germany). Samples were homogenized in TX-100 buffer and the supernatants were collected by centrifugation at 16,000g for 10 minutes. Protein concentration was determined by modified Lowry assay (Bio-Rad DC protein assay). The lysates were then loaded on 4–20% Tris-glycine gel (Invitrogen, Carlsbad, CA, USA). Protein was transferred to polyvinylidene difluoride (PVDF) membranes and incubated with protein-free T20 blocking buffer (Pierce, Rockford, IL). The membrane was incubated with antibodies against α-DG (IIH6C4) and α-actin (Sigma, St. Louis, MO, USA) in 20mM Tris pH7.4, 150mM NaCl, 0.1% Tween20 at 1:2000 dilutions. Alpha-DG and α-actin antibodies were detected by HRP-Goat anti-mouse IgM (Invitrogen, Carlsbad, CA, USA) and Goat anti-rabbit IgG-HRP conjugate (Bio-Rad, Hercules, CA, USA) respectively. Blots were developed with ECL (PerkinElmer, Waltham, MA, USA) and the images were exposed and processed by a LAS-4000 imaging system (Fujifilm, Valhalla, NY, USA).