Biotinylated goat anti mouse igg h l ba 9200

ELISA plates (Multiwell Immuno Plate, Maxisorp, 96 well; M9410, Thermo Scientific) were coated with 5 μg/ml recombinant mGal1 in PBS (pH 7.4) and blocked with horse serum. Mouse sera were diluted in 10% E. coli Rosetta gami (DE3) whole-cell extract (to reduce background) to a final dilution of 1:500. Anti-Gal1 antibodies were detected with 3 μg/ml biotinylated goat anti-mouse IgG (H + L) (BA-9200; Vector Laboratories) and 2 μg/ml streptavidin–horseradish peroxidase (SA-HRP, SA-5004; Vector Laboratories). All incubations were performed at 37 °C. HRP activity was detected with TMB substrate (T8665, Sigma-Aldrich), and absorbance was measured at 650 nm. All samples and blanks were assayed as duplicates.

ELISA plates were coated with 8 μg/ml GST-mEDA in PBS (pH 7.4) and blocked with horse serum. Mouse sera were diluted 1:125 in horse serum. Anti-ED-A antibodies were detected with biotinylated goat anti-mouse IgG (H+L) (BA-9200; Vector Laboratories, Burlingame, CA, USA) and streptavidin-horseradish peroxidase (SA-HRP; SA-5004; Vector Laboratories), both diluted 1:500. HRP-activity was detected with TMB substrate (T8665; Sigma-Aldrich) and absorbance was measured at 650 nm. All samples and blanks were assayed as duplicates. To detect anti-mED-A or anti-ED-B antibodies in sera from immunized rabbits, or after affinity purification, an ELISA was performed as described above. Serum and affinity purified antibody was diluted 1:1000 in horse serum.