Quantitative fluorescence resonance energy transfer-based immunoassay for activated complement C1s

Abstract

Objectives: C1s activation is associated with the pathogenesis of various diseases, indicating the potential value of C1s activation detection in clinic. Here we aimed to establish fluorescence resonance energy transfer (FRET)-based immunoassay for the quantitative detection of activated C1s in serum.

Methods: FRET-based fluorogenic peptides, sensitive to the enzymatic activity of activated C1s, were prepared and labeled with the fluorophore ortho-aminobenzoic acid (Abz) and quencher 2,4-dinitrophenyl (Dnp), and then were further selected depending on its Kcat/Km value. C1s in the samples was captured and separated using anti-C1s-conjugated magnetic microbeads. Next, enzymatic activity of activated C1s in samples and standards was examined using fluorescent quenched substrate assays. Limit of detection (LOD), accuracy, precision, and specificity of FRET-based immunoassay were also investigated.

Results: This method presented a linear quantification range for the enzymatic activity of activated C1s up to 10 μmol min^-1 mL^-1 and LOD of 0.096 μmol·min^-1·mL^-1 for serum samples. The recovery of the method was in the range of 90% ~ 110%. All CV values of the intra-analysis and inter-analysis of three levels in samples were less than 10%. The cross-reaction rates with C1r enzyme, MASP1, and MASP2 were less than 0.5%. No significant interferences were found with bilirubin (0.2 mg mL^-1), Chyle (2000 FTU), and haemoglobin (5 mg mL^-1), but anticoagulants (EDTA, citrate and heparin) inhibited the enzymatic ability of activated C1s. Thus, this established method can be used for the determination of active C1s in human serum samples in the concentration interval of 0.096-10.000 μmol min^-1 mL^-1.

Conclusions: One anti-C1s-based FRET immunoassay for activated C1s detection in serum samples were established, and it will be useful to explore the role of C1s activation in the pathogenesis, diagnosis and treatment in complement-related diseases.

Keywords: C1s; C2; C4; FRET; complement; immunoassay.

Figure 1

Quantitative FRET immunoassay of the activated C1s based on the capture of anti-C1s antibody: (A) MNP or Beads were precoated with antibody against activated C1s and then added into wells; (B) Activated C1s standard or samples were added and then specifically combined with MNP-labelled anti-C1s antibody; (C) Substrate peptide was added and then cleaved by activated C1s; (D) Fluorescence intensity from Abz on the substrate fragment was monitored by a microplate reader, and the enzymatic ability of activated C1s was quantitatively analysed. Abz, ortho-aminobenzoic acid; MNP, magnetic nanoparticles; FRET, fluorescence resonance energy transfer.

Figure 2

Kinetic analysis of human active C1s on cleavage of synthetic peptide substrates. Michaelis-Menten curves were generated where velocity (v_o ) was plotted against the concentrations of (A) peptide 1, (B) peptide 2, and (C) peptide 3; (D) K_cat/K_m values of the proteolytic activity of C1s on three peptide substrates. Data are presented as the means ± standard deviation (n = 3). P-values were calculated by one-way ANOVA.

Figure 3

Optimization of the reaction time and generation of standard curve. (A) The optimum reaction time was ascertained in the presence of chromogenic peptide 3 (10 μL, 1 μg/mL). Fluorescence intensity was recorded at the indicated time; (B) Plot of standard curves were generated against activated C1s with different concentrations (0.625, 1.25, 2.5, 5, and 10 μmol·min^-1·mL^-1).

Figure 4

Effects of anticoagulants on FRET-based immunoassay for activated complement C1s detection. (A) Enzyme activity of complement C1s in serum and plasma samples by FRET-based immunoassay; (B) Protein contents of C1s in serum and plasma samples by ELISA; Human blood samples (n = 18) were collected with EDTA, citrate and heparin as anti-agglutinins. (C) Activated C1s standard solutions assayed by FRET-based immunoassay after treatment with EDTA, citrate and heparin; Data are presented as the means ± standard deviation (n = 3). ns, P>0.05; **P<0.01; ***P<0.001; P<0.0001. P-values were calculated by one-way ANOVA.

Figure 5

Distribution of the activated complement C1s activity in serum from healthy individuals. The activity of C1s was analysed by FRET-based immunoassay. n = 306.

Figure 6

The levels of active C1s in serum of patients with RA. Data are presented as the means ± standard deviation (each group: n = 20). P = 0.0219 by t test.