Veronal buffer vb

As a C1q activator, human IgM (Sigma, St. Louis, MO, USA) was coated on 96-well plates at 2 µg/mL overnight at 4 °C, then blocked with 5% BSA in PBS at 37 °C for 2 h. On each well of the plates, 2 µg of C1q was added that had been pre-incubated with different amounts of rEmCRT (0, 2, 4 µg) or BSA (4 µg, as a comparison) in total volume of 100 µL for 2 h at 37 °C and incubated for 1 h at 37 °C. After washing with PBST, C1q-D diluted at 1:100 in 1 × Veronal buffer (VB, Lonza, Basel, Switzerland) containing 0.05% Tween-20 and 0.1% gelatin was added into each well for 1 h at 37 °C to finish the classical complement activation. NHS at dilution of 1:50 was used as a positive control. After being washed for three times with PBST, each well was added with 100 µL goat anti-human C4 mAb (1:1000 Abcam, Cambridge, UK) or rabbit anti-human C3 polyclonal antibodies (1:100, BOSTER Biological Technology Co., Ltd..., Chengmai, China) to determine C4 and C3 intermediate product of classical complement activation. HRP-conjugated rabbit anti-goat or goat anti-rabbit IgG (1:5000 or 1:1000, Affinity Biosciences, Liyang, China) was used as the secondary antibody and OPD (Beyotime Biotechnology, Shanghai, China) was used as the substrate.

To evaluate whether the binding of peptide P2 to C1q inhibits the C1q-initiated complement activation, the intermediate complement C4 deposition following complement activation was analyzed [29 (link)]. To initiate the classical activation, purified human IgM (Calbiochem, Merck, Darmstadt, Germany) was coated on plates (0.2 μg/well). The plates were blocked with 3% (w/v) BSA, then incubated at 37 °C for 1 h with NHS which was pre-incubated with various doses of peptide P2 (0, 2, or 6 μg) in 1× Veronal buffer (VB; Lonza, Basel, Switzerland) containing 0.05% (v/v) Tween-20 and 0.1% (w/v) gelatin. Subsequently, goat anti-human C4 mAb (1:10,000, Abcam) was added and incubated for 1 h. HRP-conjugated rabbit anti-goat IgG (1:10,000; BD Biosciences) was used as the secondary antibody. OPD was used as substrate and absorbance at 450 nm was measured.

Plates were coated with human IgM (2 µg/mL) as C1q activator and blocked with 3% BSA in PBS at 37°C for 2 h before the addition of 2 µg of C1q that had been preincubated with different doses of rTs-CRT (0, 1, or 2 µg) or BSA (2 µg). After 1-h incubation, the plates were washed and incubated with C1q-D (1:200) in 1× Veronal buffer (VB, Lonza, Switzerland) containing 0.05% Tween-20 and 0.1% gelatin for 1 h at 37°C. NHS was served as a positive control. After being washed, C4 and C3 depositions were determined using goat anti-human C4 mAb and rabbit anti-human C3b polyclonal antibodies (1:10,000, Abcam), respectively. HRP-conjugated rabbit anti-goat or goat anti-rabbit IgG (BD Biosciences) was used as the secondary antibody. The absorbance was measured at 450 nm with an ELISA reader.