Anti igm biotin

To detect the level of IgG1 and IgM antibodies in mice sera, 96 well ELISA F-bottom, clear, Microlon 600 (high binding) microplates (Greiner Bio-one) were coated with goat anti-mouse kappa-UNLB (Southern Biotech) and goat anti-mouse lambda-UNLB (Southern Biotech) antibodies at 2.5 μg/mL O/N at 4C. After washing with PBS (3X), coated plates were blocked with PBSA (PBS+0.5% BSA+0.01% azide) for 1 h at RT. Sera was added to plates at a serial dilution of 1:500, 1:1000, and 1:2000 and incubated at RT for 2 h. Plates were washed with PBS (4X) and anti-IgM-biotin or anti-IgG1-biotin (Southern Biotech) were added to wells at a final concentration of 1 μg/mL. Plates were washed with PBS (4X) and streptavidin-AP (Roche) was added to wells at 1:3000 for 1 h at RT. Plates were washed with PBS (3X) and 4-nitrophenyl phosphate (Sigma) substrate in Developing Buffer (Thermo Scientific) was added to wells. Reaction was detected by a FlexStation 3 (Molecular Devices) plate reader. Concentration of antibody in sera was determined by comparing OD to standard curve. Standard curves obtained by serial dilutions (0.025 μg/mL, 0.00625 μg/mL...9.76×10⁻⁵ μg/mL, 2.44×10⁻⁵ μg/mL) of IgM and IgG1 antibodies (Southern Biotech).