Xeroderma Pigmentosum, Complementation Group B (XPB) is a crucial DNA helicase encoded by the ERCC3 gene in humans. As a component of the transcription factor IIH (TFIIH) complex, XPB plays dual roles in nucleotide excision repair (NER) and transcription initiation. In NER, it unwinds DNA around lesion sites to facilitate damage removal, while in transcription, it helps open promoter regions for RNA polymerase II. Besides,Poly U Binding Splicing Factor 60kDa (PUF60) has been identified as an interactor of XPB, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human XPB and recombinant human PUF60 .Briefly, biotin-linked XPB were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to PUF60-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 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 recombinant human XPB and recombinant human PUF60 was shown in Figure 1, the EC50 for this effect is 0.29 µg/mL.