Individual complement analytes were evaluated using a modified MILLIPLEX MAP Human Complement Panel 1 and 2 (MilliporeSigma, Supplemental Table 3 ) based on the Luminex xMAP technology, a bead-based multiplex assay. Specifically, we used the MILLIPLEX MAP Human Complement Panel 1 (HCMP1MAG) to simultaneously quantify the following analytes in the BAL: complement C2, complement C4b, complement C5, complement C5a, complement C9, adipsin (complement factor D), and MBL. Of note, this assay is specific for C4b and does not cross-react with C4d (per communication with manufacturer). The C5 measurements are distinct from C5a, as the intact factors are designed such as they would not detect individual fragments based on their capture and detection antibodies.
Similarly, we used the MILLIPLEX MAP Human Complement Panel 2 (HCMP2MAG) to simultaneously quantify the following analytes in the BAL: complement C1q, complement C3, complement C3b/iC3b, complement C4, and complement factor B. Of note, the assay for C3b/iC3b detects both C3b and iC3b (per communication with manufacturer). The factor B assay does not detect either fragments Bb or Ba.
Similarly, we used the MILLIPLEX MAP Human Complement Panel 2 (HCMP2MAG) to simultaneously quantify the following analytes in the BAL: complement C1q, complement C3, complement C3b/iC3b, complement C4, and complement factor B. Of note, the assay for C3b/iC3b detects both C3b and iC3b (per communication with manufacturer). The factor B assay does not detect either fragments Bb or Ba.