IRAS is a large protein comprising of 1504 amino acids. Its NH 2 -terminal phox domain is important for membrane association and cellular localization. The N-terminal of IRAS (10-120aa) covering the PX domain was cloned into the pET-43.1a(+) (Figure 1(a) ) [29] . The supernatant fusion protein was purified by His-tag affinity purification and was subsequently used to generate the monoclonal antibody. The dissolved protein yielded one major band of 78 kDa expected molecular weight (Figure 1(b) ) with high purity and integrity. The NusA protein used as a control generated the 66 kDa expected molecular weight. The recombinant protein was also confirmed with western blot using anti-His mAbs (right column, Figure 1(b) ). BALB/c mice (n = 3) were immunized with the NusA-IRAS (10-120aa) fusion protein, and blood was collected from the mice after multiple injections. Antibody titers were tested by ELISA on plates coated with the NusA-IRAS (10-120aa) protein (data not shown). The 2 mice with the highest titers were sacrificed and spleens from both mice were fused to myeloma cells following standard procedures. Individual hybridomas was grown and 125 hybridomas were further characterized. Supernatant from the growing hybridoma clones was screened with ELISA. Screening was performed on plates coated with NusA-IRAS (10-120aa) , NusA protein, and GST-IRAS (10-120aa) fusion protein to determine antibody specificity. A total of 5 hybridomas (DA041, DD015, BE073, BA022, and AH021) reacted selectively with the NusA-IRAS (10-120aa) protein in all 3 assays and were further evaluated. Isotype analysis revealed that all mAbs were of the IgG1 subtype.