Cathepsin V (CTSV) is a lysosomal cysteine proteinase which belongs to peptidase C1 family. It is expressed in normal human thymus, testis and corneal epithelium. Cathepsin V plays an important role in corneal physiology and mediates degradation of invariant chain in human thymus. Besides, Retinoblastoma Protein 1 (RB1) has been identified as an interactor of CTSV, thus a binding ELISA assay was conducted to detect the interaction of recombinant human CTSV and recombinant human RB1. Briefly, CTSV were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to RB1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-CTSV pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of CTSV and RB1 was shown in Figure 1, and this effect was in a dose dependent manner.