C1qB protein is a component of the C1 complex in the classical pathway of the complement system. It is a subunit of the C1q molecule, which is the first component of the complement system to initiate the classical pathway. C1q is a hexameric protein composed of six identical subunits, each containing a C1qB, a C1qB, and a C1qC subunit. C1qB plays a key role in eliminating pathogens, regulating inflammatory response and maintaining immune tolerance. The binding of C4b and C1qB is a key step in the activation of the classical pathway in the complement system, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human C1qB and recombinant mouse C4b. Briefly, C1qB was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to C4b-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-C1qB pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, 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 450/630 nm immediately. The binding activity of recombinant human C1qB and recombinant mouse C4b was shown in Figure 1, the EC50?for this effect is 0.12 ug/mL.