ELISA methods for S. suis

Antibodies to serotype 2 S. suis were measured using an enzyme-linked immunosorbant assay (ELISA). The binding of sera to whole S. suis cells (serotype 2, strain 30-336-06),was detected by inoculating strain into THB and growing at 37°C until the optical density (O.D.) reached approximately 1.2. The bacteria were then pelleted and washed once in PBS and resupsended to an O.D. of 1.0. The cell suspension was then diluted 1:32 in a 50mM carbonate-bicarbonate coating buffer and 100 µL was added to microtiter wells, which were incubated at 37°C for 1 hour and overnight at 4°C. The plates were then washed 3X with 150 µl of wash buffer (PBS/0.05% Tween-20). Anti-S. suis antibody (Statens Serum Institut, Copenhagen, Denmark) was added as a positive control and serial 2-fold titrations made in 0.1% BSA/PBS from 1:500 to 1:64,000. Dilutions of sera samples were made in 0.1% BSA/PBS and serial 2-fold titrations made from 1:10 to 1:1,280. All samples were run in duplicate. Plates were incubated for 2 hours at 37°C. Plates were then washed 3X with 150 µl of wash buffer. Unbound sites were blocked with BSA and incubated with a 1:15,000 dilution of recombinant protein A, horseradish peroxidase conjugated (Pierce Biotechnology, Inc., Rockford, IL) in Wash Buffer for 2 hours at room temperature. The plates were then washed 3X with 150 ul of wash buffer and developed with TMB Microwell Peroxidase Substrate System (2-C) (KPL, Gaithersburg, Maryland). Optical density was read at 650nm using microplate reader Versamax (Molecular Devices, Sunnyvale, California) with SoftMax Pro software (Molecular Devices). Plates were rejected if the positive controls varied more than 2 standard deviations from the mean value determined during assay optimization.