Fig. 1

Characterization of FHR-2 monoclonal antibodies (mAbs). (a) An overview illustrating the genetic similarity between FHR-1, -2, and FHR-5. (b) Cross-reactivity analysis of the αFHR-2 mAbs. Wells were coated with the indicated αFHR-2 mAb and incubated with biotinylated plasma-derived FH (pdFH) or recombinant human FHR (rhFHR) proteins (10 nM). Current non-specific αFHR-2 mAbs (αFHR-2.1, αFHR-2.4) and an irrelevant isotype mAb (neg. contrl.) were included as controls¹¹. Absorption levels above 0.1 (dotted line) were considered indicative of cross-reactivity. Some test conditions resulted in absorption levels exceeding the upper limit of quantification, set at 3.0. (c–e) Western blot analysis of immunoprecipitation using αFHR-2.11 to αFHR-2.15 in normal human serum (c), CFHR3/CFHR1 deficient serum (determined via MLPA) (d), and serum deficient for FHR-2 (previously determined via gene sequencing) (e)¹¹. All three immunoblots were stained using αFHR-2.1 (cross-reactive for FHR-1 and -2) to verify specificity for native FHR-2 and exclude cross-reactivity for native FHR-1. (f) Competition ELISA including selective αFHR-2 mAbs. Biotinylated rhFHR-2 (0.1 µg/mL) was pre-incubated with αFHR-2 mAbs (10 µg/mL) for twenty minutes before adding to the ELISA plates coated with indicated mAbs. Binding of biotinylated rhFHR-2 is expressed as relative binding of biotinylated rhFHR-2 in the presence of an isotype control. Bars represent the mean of two independent replicates with error bars indicating the SD. ELISAs and Western blots are representatives of three independent experiments.