1 step turbo tmb

LOS samples were analyzed following a standardized ELISA protocol. Briefly, Nunc Maxisorp plates (Thermo) were coated with 50 μL of purified LOS and incubated at 37°C for 1 hour. Plates were washed twice with 300 μL of wash buffer using a BioTek ELx50 plate washer. Wells were then blocked with 200 μL 0.5% bovine serum albumin in 1X DPBS at 37°C for 1 hour. Plates were washed again and the LOS was probed with 50 μL of a 1:200 dilution of either anti-Lipid A antibodies (Abcam Cat# ab8467, RRID:AB_306573) or chinchilla serum. Samples were incubated for 1 hour and then washed 3 times. 50 μL of a 1:3000 dilution of anti-Ms IgG-HRP (Abcam Cat# ab205719, RRID: AB_2755049) or HRP-Protein A (Invitrogen 101023) were used to detect Lipid A and chinchilla antibodies, respectively. Samples were incubated for 1 hour and then washed 3 times as described previously. 1-Step™ Turbo TMB (100 μL, Thermo 34022) was added to each well to visualize the HRP conjugated antibodies. The reaction in wells containing Lipid A + Goat pAb to Ms IgG was stopped with 100 μL 2M H₂SO₄ after 2 minutes. Wells containing chinchilla post-serum + Protein A-HRP were stopped with 100 μL 2M H₂SO₄ based on the oafA OFF samples, up to 5 minutes. Absorbance at 450 nm was measured with an H1MG plate reader (BioTek).