Igm or igg antibodies

96-Well plates (Nunc, ThermoScientific) were coated either with 10 µg/ml anti-human IgM or anti-human IgG-antibodies (SouthernBiotech) or with 10 µg/ml human IgG (SouthernBiotech) or with 2,5 µg/ml calf thymus dsDNA (Rockland) or with 2,5 µg/ml native insulin (Sigma-Aldrich). Blocking was done in 1% BSA blocking buffer (SERVA). Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard. The relative concentrations stated as arbitrary unit (AU), were determined via detection by alkaline phosphatase (AP)-labeled anti-IgM/anti-IgG (SouthernBiotech). The p-nitrophenylphosphate (pNPP; Genaxxon) in diethanolamine buffer was added and data were acquired at 405 nm using a Multiskan FC ELISA plate reader (Thermo Scientific). All samples were measured in duplicates.
Antibody specificity, host/isotype, conjugate clone, class, supplier catalog number: anti-human IgM (goat, IgG, unlabeled, polyclonal, SouthernBiotech, #2020-01); anti-human IgG (goat, IgG, unlabeled, polyclonal, SouthernBiotech, #2040-01); human IgM (unlabeled, SouthernBiotech, #0158L-01); human IgG (unlabeled, SouthernBiotech, #0150-01); anti-human IgM (mouse, AP, monoclonal, SouthernBiotech, #9020-04); anti-human IgG (Goat, AP, polyclonal, SouthernBiotech, #2040-04).

96-Well plates (Nunc, Maxisorp) were coated with, native Insulin (Sigma-Aldrich, Cat. 91077C), Streptavidin (ThermoScientific, Cat. 21125), calf thymus DNA (ThermoScientific, Cat.15633019), or NP-BSA (Biosearch Technologies, N-5050H-100) with 10 µg/mL, or anti-IgM, anti-IgG-antibodies (SouthernBiotech). Biotinylated peptides (2,5 µg/mL) were loaded onto SAV-coated plates in 1% BSA blocking buffer (ThermoFisher). Sera were initially diluted 1:50. Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standard. The relative concentrations, stated as arbitrary unit (AU), were determined via detection by Alkaline Phosphatase (AP)-labeled anti-IgM/anti-IgG (SouthernBiotech), respectively. The p-nitro-phenylphosphate (pNPP; Genaxxon) in Diethanolamine buffer was added for starting of the reaction. Data were acquired at 405 nm using a Multiskan FC ELISA plate reader (Thermo Scientific). Samples were measured at least in duplicates.
For analysis of affinity-maturation (22 (link)), results from plates coated with InsA(1) or InsA(4) were calculated by dividing InsA(1) by InsA(4). Here, we used streptavidin with tetra- (ThermoScientific) or monovalent binding capacity. Subsequently, results were stated as relative units [RU].