Fig 1.
Characterisation of anti-FHR-3 mAbs.
(A) Cross-reactivity of the obtained anti-FHR-3 monoclonal and polyclonal Abs. Wells coated with the indicated Abs were incubated with 10 nM of biotinylated FH, rhFHR proteins or an irrelevant 6xHis-tagged protein as control. Binding was determined by ELISA. (B) IP of FHR-3 from serum by anti-FHR-3.1 and anti-FHR-3.4 mAbs, visualized by Western Blot using poly anti-FHR-3-bt. Left panel; IP from healthy donor serum with two CFHR3 gene copies. Right panel; IP from healthy donor serum with no CFHR3 gene copies present (determined by MLPA). (C) IP of FHR-3 with anti-FHR-3.4 from a CFHR3 sufficient donor, stained with anti-FHR-3.1-bt. (D) IP and staining of FH, FHR-3, FHR-4A and FHL-1 on Western blot from healthy donor plasma using poly anti-FHR-3, prior or after depletion with either anti-FHR3.1 or anti-FHR-3.4.
Table 1.
CFHR3/CFHR1 copy number variation in 100 healthy donors.
Fig 2.
Characterisation of the FHR-3 ELISA with anti-FHR-3.1 and anti-FHR-3.4.
(A) Titration of 5 μg/ml rhFHR-3, pool serum and serum of three donors with known copy numbers for CFHR3 in the FHR-3 ELISA. Representative of three independent measurements, points represent the mean with error bars indicating SD of duplicates. (B) Influence of additional rhFHR-4A on FHR-3 levels measured in the FHR-3 ELISA. Pooled serum was incubated with excess rhFHR-4A, as indicated, prior to measuring the FHR-3 levels in the FHR-3 ELISA. Representative of two independent measurements, points represent the mean with error bars indicating SD of duplicates. (C) FHR-3 levels in serum and plasma as measured with the FHR-3 ELISA in 7 healthy donors. Points represent means of three independent measurements per donor. Wilcoxon paired test; NS = not significant.
Fig 3.
FHR-3 levels correlate with gene copy numbers and do not show an early acute phase response.
(A) FHR-3 serum levels were measured by ELISA in 100 healthy donors and arranged by CFHR3 gene copies as identified by MLPA. (B) FH serum levels were measured by ELISA in the same donors and depicted in relation to FHR-3 levels. The four donors with no copies of CFHR3 (open symbols) were excluded from the correlation analysis (Spearman’s test). (C) FHR-3 levels were measured by ELISA in 39 acute sepsis patients and compared to FHR-3 levels of the healthy donor cohort depicted in A. (D) Graph depicting the correlation between FHR-3 levels and CRP levels. The two sepsis patients with apparent CFHR3 deficiency (open symbols), as no FHR-3 was detectable, were excluded from the correlation analysis (Spearman’s test). Points in A-D represent the means of three independent measurements per donor or patient. Lines in A and C represents the mean with error bars indicating the 95% CI. Data were analyzed using the Kruskal-Wallis test (A) or Mann-Whitney test (B); NS = not significant, * p<0.05, *** p<0.001, **** p<0.0001.