Gelatin veronal buffer

CVF is a stable anti-complement protein and functionally resembles C3 purified from Cobra venom. To testify complement depletion induced by CVF pretreatment, complement activities were determined by the 50% haemolytic complement (CH50) activity in serum and the C3 level in the plasma and BALF. The CH50 is a screening assay which can test the functional capability of serum complement components. The level of CH50 in serum was determined by hemolytic assay^{17 (link)}. In brief, serum samples were sensitized with goat anti-serum and incubated at 30 °C for 60 min to serial dilutions in gelatin veronal buffer (Sigma, St. Louis, MO, USA). After a centrifugation step (2500 g, 5 min), the supernatant fluid was collected and measured at 405 nm. The C3 levels in the plasma and BALF were determined by ELISA (catalogue number: ab157731; Abcam, Cambridge, MA) according to the manufacturer’s instructions. The quantity of C3 was standardized to a predetermined standard quantity derived from SD rat.

Immune complexes were formed by adding 10 µL of blue-coupled beads to 96-well u-bottom plates and incubating with 10 µL diluted purified mAb or clinical sample for 2 hours at 37°C and 5% CO₂. After incubation, the immune complexes were washed with RPMI medium (Gibco, 21870076). Guinea pig serum (Sigma, 234395) was diluted 1:60 in gelatin veronal buffer (Sigma, G6514), added to the plates, and incubated for 50 minutes at 37°C and 5% CO₂ to facilitate binding between complement proteins and anti-RSV F protein immune complexes. The reaction was then stopped by washing the plates twice with 15 mM ethylenediaminetetraacetic acid in PBS. To detect complement deposition, plates were incubated with fluorescein isothiocyanate-conjugated goat anti-guinea pig complement C3 antibody (MP Biomedicals, 0855385) for 20 minutes in the dark. Plates were washed with PBS and fluorescence was measured on a BD LSRFortessa. The results were reported as the average (n = 3 technical replicates) median fluorescent intensity.

ADCD was determined by the deposition of the complement component C3b on the surface of CEM-NK_R.CCR5 cells as described in [10 (link)]. Target cells were pulsed with 10μg BG505.SOSIP.664 gp140 trimer in 100μl of R10 media (10% FBS 1% Pen/Strep RPMI, Gibco, Gaithersburg, MD) for 1 hour at room temperature and incubated with 100μg/ml of IgG preparation. HIV-negative plasma was used as a source of complement and diluted 1 in 10 in 0.1% gelatin/veronal buffer (Sigma-Aldrich, St Louis, MO) and 150μl added and incubated for 20 minutes at 37°C. The cells were then washed in 15mM EDTA in PBS and C3b was detected by flow cytometry using an anti-human/mouse complement component C3/C3b/iC3b mAb (Cedarlane, Burlington, Canada). Unpulsed cells were used as background controls and HIV-negative plasma was heat-inactivated at 56°C to remove complement as a negative control. The ADCD score was defined as geometric MFI multiplied by % cells positive for C3b deposition.

Alternative complement activity (ACH50) was measured based on [82 ]. In brief, sheep red blood cells and gelatin veronal buffer (Sigma, USA) were applied. Each 20-μL aliquots of a serially diluted serum with ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid -Mg2+-gelatin veronal buffer (GVB) as a complement source was introduced into 6 μL of sheep red blood cells (SRBC)suspension (4 × 108 cells ml⁻¹), and later they were incubated at 21 °C and pH 7.2 for 2 h. Two hundred μL of GVB containing 10 mM Ethylenediaminetetraacetic acid (EDTA) was added into the suspension to stop the hemolytic activity. The suspensions were centrifuged at 1600× g for 10 min at 4 °C and OD was read at 414 nm using an ELISA microplate reader. The reactions were supplemented with 6 μL EDTA-GVB, 20 μL EDTA-GVB, and 220 μL distilled water to replace the SRBC suspension, the diluted mucus sample, and the diluted mucus + EDTA-GVB, respectively, as the SRBC blank, serum blank, and 100% hemolysis sample. The ACH50 activity level of hemolysis was calculated for all treatments.

S. pneumoniae ATCC6303 was grown on blood agar plates for 18h. Bacteria were diluted in THY, grown until OD_{600 nm =} 0.4–0.5 (~10⁸ CFU/mL) and harvested by centrifugation at 3,200 x g for 10 min. Bacteria were then washed, suspended in PBS and incubated with 5% (V/V) of sera from immunized mice (pooled sera from each experimental group) during 30 min on ice. Samples were then washed once with PBS before incubation for 30 min with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (MP Biomedicals, California, USA), diluted 1:100 in PBS. For complement deposition assays, sera were previously inactivated at 56°C for 30 min and incubated with bacteria at a concentration of 10 or 20%, for 30 min on ice. Samples were washed once with PBS and incubated with 10% of normal mouse serum as source of complement, in Gelatin Veronal buffer (Sigma Aldrich), at 37 ºC for 30 min. After washing, samples were incubated with FITC-conjugated anti-mouse C3 IgG (MP Biomedicals) in PBS, for 30 min on ice. In both experiments, samples were fixed with 200 μL of cytofix (BD Biosciences, California, USA) after two washing steps. Flow cytometry analysis was conducted using FACS Canto II (BD Biosciences), and 15,000 gated events were recorded. Fluorescence was analyzed by histograms using the Flow Jo 10.1 software and medians of the curves were used to compare the groups.