Fig. 2

ELISA validation and calibration. (a) Selection of monoclonal antibody (mAb) sandwich combination to enable detection of FHR-2/2 homodimers in normal human serum (NHS). Combinations of the same mAb (bolt bordered squares) are of interest based on the principal only a homodimer presents a single FHR-2 epitope twice. ELISA plates were coated with a FHR-2 specific αFHR-2 mAb and incubated with 20% (v/v) NHS. Next, bound FHR-2 was detected using indicated biotinylated αFHR-2 mAb. Absorption levels above 0.1 were considered indicative of antigen detection. Validation of FHR-2/2 (b) and FHR-1/2 (c) assay specificity using NHS and serum deficient for FHR-1 or -2. The FHR-2 specific αFHR-2.11 mAb was coated on ELISA plates and incubated with either NHS and serum deficient for FHR-1 or -2 (4%, (v/v)). Next, bound target protein was detected using biotinylated αFH.02 (αFH mAb cross-reactive with FHR-1) or αFHR-2.11 for FHR-1/2 and -2/2 dimers, respectively²³. (d) FHR-2/2 assay calibration of an NHS standard using rhFHR-2. For the calibration of the FHR-1/2 ELISA, seven mixes with different volumetric ratios (mix A: 95–5%, B: 85–15%, C: 75–25%, D: 50–50%, E: 25–75%, F: 15–85%, G: 5–95% (v/v)) of a plasma deficient in FHR-2 or -1 respectivaly were mixed and incubated at 37 °C for six hours to allow the formation of FHR-1/2 heterodimers. (e) NHS standard pool calibrated for FHR-1/2 using each mix as standard. FHR-1/2 levels in each mix were based on the average decline in FHR-1/1 and -2/2. Dotted line represents the mean of mix C-G with the grey area indicating the mean ± 15%. (f) Levels of all three FHR-1 and -2 dimer species in each mix (start concentration FHR-1/1: 273.84 nM; start concentration FHR-2/2: 81.20 nM) after incubation at 37 °C. (a–d) n= 3, representative is shown. (b–f) Symbols represent mean of multiple measurements with error bars indicating the SD.