Fig 1.
Prokaryotic expression and purification of CCHFV-NP.
(A) Primary structure of recombinant CCHFV-NP fusion protein encoded by the expression vector pOPINJ-CCHFV-NP (6xHis-GST-3C-CCHFV-NP). The full-length CCHFV-NP is N-terminally tagged with both a 6 x His-tag and GST. A 3C protease recognition site enables tag removal after purification. (B) Induction of protein expression by IPTG. Protein expression was induced by IPTG in E.coli pAPlacIQ cells transformed with pOPINJ-CCHFV-NP. Total bacterial lysates (pre: before induction, post: after induction) were analyzed by SDS-PAGE followed by Coomassie-staining. Arrow head: 6xHis-GST-3C-CCHFV-NP (calculated molecular weight: 82 kDa). (C) Ni-NTA affinity purification of 6xHis-GST-3C-CCHFV-NP under native conditions. E.coli pAPlacIQ harbouring 6xHis-GST-3C-CCHFV-NP were lyzed by lysozyme and sonification. After centrifugation, the soluble supernatant (SN) was applied to Ni-NTA agarose. Bound material was eluted by imidazole. FT: flow through, eluates: E1, E2, E3. Arrow head: 6xHis-GST-3C-CCHFV-NP (calculated molecular weight: 82 kDa). (D) Removal of 6xHis-GST tag by on-column 3C protease digest. Eluates from Ni-NTA affinity purification were pooled (In: Input) and applied to Glutathion HiCap matrix (FT: Flow-through). On-column cleavage was performed using recombinant, GST-tagged 3C-protease. E: Eluate from matrix (CCHFV-NP without tag, calculated molecular weight: 54 kDa), M: Proteins attached to the matrix after elution.
Fig 2.
Detection range of BLACKBOX CCHFV IgM/IgG ELISAs.
(A) BLACKBOX CCHFV IgM ELISA. A CCHF patient serum (IIFT IgM titer 1: 640) was spiked into a negative human serum at different dilutions (1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512) and analyzed with the BLACKBOX CCHFV IgM ELISA. In parallel, 71 CCHFV IgM negative sera from healthy human blood donors (n = 49) and malaria infected (n = 22) patients were tested (mean optical density of signals obtained for CCHFV IgM negative sera: 0.032, standard deviation: 0.011). (B) BLACKBOX CCHFV IgG ELISA. A CCHF patient serum (IIFT IgG titer 1: 10240) was spiked into a negative human serum at different dilutions (1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400) and analyzed with the BLACKBOX CCHFV IgG ELISA. In parallel, 71 CCHFV IgG negative sera from healthy human blood donors (n = 49) and malaria infected (n = 22) patients were tested (mean optical density of signals obtained for CCHFV IgG negative sera: 0.072, standard deviation: 0.017).
Table 1.
Intra- and interassay variation of BLACKBOX CCHFV IgM ELISA.
Table 2.
Intra- and interassay variation of BLACKBOX CCHFV IgG ELISA.
Table 3.
Inter-laboratory variation of BLACKBOX CCHFV IgM ELISA.
Table 4.
Inter-laboratory variation of BLACKBOX CCHFV IgG ELISA.
Fig 3.
Analysis of paired CCHF patient samples: Raw data (OD420 – OD620).
A serum panel consisting of 30 paired serum samples from 15 CCHF patients, serum samples from 12 CCHF patients collected approximately one year after recovery from CCHFV infection, a set of a priori CCHFV IgM/IgG negative serum samples (neg) ((A), (B): n = 120; (C), (D): n = 15, see S1 Fig), and 98 CCHFV IgM/IgG IIFT negative sera from healthy blood donors from Kosovo (HD K) were analyzed with the BLACKBOX CCHFV IgM ELISA (A), the BLACKBOX CCHFV IgG ELISA (B), the VectoCrimean-CHF-IgM ELISA (C) and the VectoCrimean-CHF-IgG ELISA (D). Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see S2 Fig). Filled circles indicate PCR positive serum samples.
Table 5.
CCHF patient sera information and results summary.
Table 6.
Analysis of paired CCHF patient samples: Comparison of BLACKBOX CCHFV IgM ELISA, VectoCrimean-CHF IgM ELISA and in-house IgM IIFT results.
Table 7.
Analysis of paired CCHF patient samples: Comparison of BLACKBOX CCHFV IgG ELISA, VectoCrimean-CHF IgG ELISA and in-house IgG IIFT results.
Fig 4.
Analysis of paired CCHF patient samples: IgG/IgM ratio.
Ratios of OD450 – OD620 values obtained with the BLACKBOX CCHFV IgG/IgM ELISA (A) and the VectoCrimean-CHF-IgG/IgM ELISA (B) were calculated for the analyzed serum samples. Filled circles indicate PCR positive serum samples. Statistical testing (One-way ANOVA/Tukey’s multiple comparison test) was performed with GraphPad Prism (ns: not significant (p > 0.05); **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).
Fig 5.
Analysis of CCHF patient samples: Raw data (OD450 – OD620), dependence from day after onset.
(A) BLACKBOX CCHFV IgM ELISA. (B) BLACKBOX CCHFV IgG ELISA. (C) VectoCrimean-CHF-IgM ELISA. (D) VectoCrimean-CHF-IgG ELISA. Paired serum samples from 15 CCHF patients were classified according to their time point of collection (days after onset of symptoms). Filled circles indicate PCR positive serum samples. Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see S2 Fig).
Table 8.
Analysis of CCHF patient samples, dependence from day after onset: Comparison of BLACKBOX CCHFV IgM ELISA, VectoCrimean-CHF IgM ELISA and in-house IgM IIFT results.
Table 9.
Analysis of CCHF patient samples, dependence from day after onset: Comparison of BLACKBOX CCHFV IgG ELISA, VectoCrimean-CHF IgG ELISA and in-house IgG IIFT results.
Fig 6.
Analysis of CCHF patient samples: Raw data (OD450 – OD620), correlation with IIFT titer.
(A) BLACKBOX CCHFV IgM ELISA. (B) BLACKBOX CCHFV IgG ELISA. (C) VectoCrimean-CHF-IgM ELISA. (D) VectoCrimean-CHF-IgG ELISA. 42 serum samples from 15 CCHF patients were classified according to their CCHFV IgM (A, C) and IgG IIFT titer (B, D), respectively. IIFT was performed using acetone-fixed Vero cells infected with CCHFV. Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see S2 Fig). Time of sampling (days after onset of symptoms) is indicated by the assigned symbols.