Abstract: Objective:To develop the hybridoma cells secreating the monoclonal antibody (McAb) against Mycobatcteium tuberculosis heat-resistant antigen (Mtb-HAg),and identify the specificity of the McAb and its biological activity.Methods:BALB/C mice were immunized 3 times with Mtb-HAg,and the spleen cells of immunized mice were fused with myeloma cells SP2/0 at the ratio of 10:1 by 50% PEG,and cultured in the HAT medium,followed by selection and subclone procedure by routine limited dilution method.The specificity and titer of the McAb were detected by ELISA method.Results:After fused 2 times,and selected and cloned 3 times,three hybridoma cell lines,Ⅰ C12,ⅡE4,and Ⅱ G8 were generated,and titers of their McAb were 1:32 000,116 000,and 116 000,respectively.ⅠC12 only had positive reaction with the Mtb-HAg protein peak Ⅰ,whereas ⅠC12 had no inhibitory effect on the Mtb-HAg activity to stimulate human γδ T cells.ⅡE4 andⅡG8 showed no positive reaction with Mtb-HAg protein peak Ⅰ,peak Ⅱ and peak Ⅲ.Conclusions:By the hybridoma technique,three hybridoma cell lines have been generated and the hybridoma Ⅰ C12 cells produce the specific McAb against Mtb-HAg protein peak I.