MBL2 (Mannose-binding protein C) is a calcium-dependent lectin involved in innate immune defense, which binds mannose, fucose and N-acetylglucosamine on different microorganisms, therefore results in activation of the lectin pathway of the complement system. It has been proven that MASP-2 (Mannan-binding lectin serine protease 2) forms complexes with the pattern recognition molecules MBL2, triggers the activation of the complement system. Thus, a functional binding ELISA assay was constructed to detect the association of rhMBL2 with MASP2. Briefly, rhMBL2 were diluted serially in 10mM Tris-HCl, 1M NaCl, 5mM CaCl2, and 0.05%Triton X-100 (pH 7.4). Duplicate samples of 100uL were then transferred to MASP2-coated microtiter wells and incubated for 2h at 37oC. Wells were washed with PBST and incubated for 1h with anti-MBL2 mAb, then aspirated and washed 3 times. After incubation with HRP labeled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution , wells were incubated for 15-25 minutes at 37oC. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of MBL2 with MASP2 was shown in Figure 1 and this effect was in a dose dependent manner.
Figure 3. SDS-PAGE
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