PBMCs were separated from blood on Ficoll-Paque by differential centrifugation and were suspended in 24-well tissue culture plates culture medium. Different dilutions of PBMCs  were incubated at 37 °C with 5% . Culture supernatants were collected at 24, 48, 72, and 96 h after incubation and the supernatants were test against BCG or PPD by ELISA. The ELISA titer indicate the positive or negative result.