R-Spondin 1 (RSPO1, Roof plate-specific Spondin 1), also known as cysteine-rich and single thrombospondin domain containing protein 3 (Cristin 3), is a 27 kDa secreted protein that shares~40% amino acid (aa) identity with three other R-Spondin family members. R-Spondin 1 competes with the Wnt antagonist DKK-1 for binding to the Wnt co-receptors, Kremen and LRP-6, reducing their DKK-1-mediated internalization. Postnatally, R-Spondin 1 is expressed by neuroendocrine cells in the intestine, adrenal gland and pancreas, and by epithelia in kidney and prostate. Besides, Leucine Rich Repeat Containing G Protein Coupled Receptor 5 (LGR5) has been identified as an interactor of RSPO1, thus a binding ELISA assay was conducted to detect the interaction of recombinant human RSPO1 and recombinant mouse LGR5. Briefly, biotin-linked RSPO1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to LGR5-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 50 µl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant human RSPO1 and recombinant mouse LGR5 was shown in Figure 1, the EC50 for this effect is 0.099 ug/mL.