Proclin 300

Information on the source of all Abs used in the SBA can be found in table S1. HPA Abs from the HPA are commercially available from Atlas Antibodies AB. HEK293 FreeStyle cells (HEK293F), FreeStyle 293 Expression media, FreeStyle MAX Reagent, and 125-ml culture flasks were from Thermo Fisher Scientific. PE-conjugated HA and FLAG Abs, as well as unconjugated HA Ab, were from BioLegend. 1D4 and OLLAS Abs were conjugated to PE using an Ab conjugation kit from Abcam (Cambridge, MA) according to the manufacturer’s protocols. Half-volume, 96-well plates were from Greiner (Tucson, AZ). Blocking reagent for ELISA (enzyme-linked immunosorbent assay) (BRE) and cOmplete protease inhibitor tablets were from Roche (Basel, Switzerland). Phosphate-buffered saline (PBS) was from Medicago. ProClin 300, casein, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and FLAG M2 Ab were from Sigma-Aldrich (St. Louis, MO). n-Dodecyl-β-d-maltoside (DM) was from Anatrace. Rabbit IgG was from Bethyl. Expression constructs of human GPR182 and GPR4 were obtained from cDNA.org. Anti-mouse IgG– and anti-Rabbit IgG–conjugated R-PE were from Jackson ImmunoResearch.

The Vn96 peptide (PSQGKGRGLSLSRFSWGALTLGEFLKL) was previously developed in our laboratory [26 (link)]. The peptide was prepared at 2.5 mg/mL in peptide resuspension buffer [0.625× PBS + 0.05% Pro-Clin300 (cat # 48912-U, Sigma,St.Louis, MO, USA)]. Precipitation of EVs and cf-DNA from EDTA plasma was performed by diluting 1 mL of plasma in 1 mL of 1× PBS and then adding 0.25% (v/v) protease inhibitor cocktail (cat# 539134, Cedarlane, Burlington, ON, Canada)) and 0.1 mg/mL of Vn96 peptide. The Vn96/plasma solution was rotated end-over-end for 1 h at room temperature and then centrifuged at 17,000× g for 15 min to pellet the peptide/EV/cf-DNA complexes. The pellet was washed 3 times with 1× PBS followed with centrifugation at 17,000× g for 10 min between washes. To control for non-specific precipitation, a vehicle control consisting of an equivalent volume of peptide resuspension buffer was included in western blotting experiments.

Antibodies were diluted in buffer and immobillized on carboxylated magnetic beads with different IDs (Luminex Corp.) as described previously16; 3 of each serum sample was transferred to microtiter plates and labelled with biotin. The labelling reaction was stopped by adding 0.5 M Tris‐HCL, pH 8.0.16 The samples were subsequently heat treated, and 1  was diluted in 50 of assay buffer consisting of PBS‐T 0.05%, 10% v/v rabbit IgG, and 1:1000 ProClin™300 (Sigma Aldrich) and incubated overnight at room temperature with the generated antibody bead array. For detection of captured proteins, the beads were washed and incubated with R‐phycoerythrin conjugated streptavidin (Invitrogen). After washing the beads, raw median fluorescent intensity (MFI) and the total bead count was recorded for each target analysed in each sample16 in a Luminex FM3D instrument (Luminex Corp.). Raw values were normalized using probabilistic quotient normalization method28, 29 prior to further analysis.

An aliquot of TTX-coupled affinity resin obtained as above was packed in a glass column (1 × 3 cm) and washed with 100 mL PBS(−). The precipitate obtained by 50% saturated ammonium sulfate fractionation from 7.5 mL portion of antiserum (Rabbit No.3, exsanguinated 2 weeks after the final inoculation) was dissolved in 7.5 mL PBS(−), loaded on the affinity column, and treated with 60 mL PBS(−) and 100 mL of 0.1 M glycine-HCl buffer (pH 2.7), successively, at room temperature. Each 2 mL fraction eluted with 0.1 M glycine-HCl buffer was collected in test tubes added beforehand with 400 L of 1 M 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), while monitoring the absorption at 280 nm using a spectrophotometer (V-550, Jasco). Isolated antibody specific for TTX and its analogs thus obtained was mixed with an equivalent volume of PBS(−) containing 6 L ProClin 300 (Sigma-Aldrich, 48912-U), divided to each 1 mL, and stored in a deep freezer (−80 °C) until use.

Each inactivated virus preparation was pre-titrated to determine optimal working dilution by flaviMIA testing of serial two-fold dilutions against known positive human sera and selecting the dilution giving maximum reactivity. Each virus preparation was diluted to 4 mL in MIA buffer (PBS + BSA (Sigma-Aldrich, USA) + ProClin300 (Sigma-Aldrich, USA) as a preservative) then mixed with its corresponding 6B6C-1-coupled beadset at 1:20. The virus–bead mixes were stored in light-resistant 4 mL bottles (Amber bottles, Nalgene, Rochester, NY, USA) which were incubated at room temperature on a shaker for several hours after which they were stored at 4 °C.

ACE-2 recombinant protein and RBD recombinant protein were purchased from OkayBio (Nanjing, China). Polyvidone (PVP), sucrose, ProClin™ 300, and bovine serum albumin were obtained from Sigma-Aldrich (Germany). N-Hydroxysulfosuccinimide sodium salt (NHS), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), polyethylene glycol (PEG-20,000), and TWEEN-20 were purchased from Aladdin (Shanghai, China). Reference material of Nab with purity higher than 95% was provided by Suzhou novoprotein (Suzhou, China). 2-Morpholinoethanesulfonic Acid (MES) was obtained from TCI (Shanghai, China). 400 nM Carboxylated LMs (Blue/Red) were provided by Magsphere (Pasadena, CA, USA). Ultrapure water (18 MΩ cm at 25 °C) purified with a Milli-Q system from Millipore Corp. (Bedford, MA, USA) was used for solution preparation.
UniSart CN 140 nitrocellulose membranes were obtained from Sartorius (Shanghai, China). Glass fiber Pads SB-08, XYZ 3D film spraying instrument, CNC cutting machine (CTS300), and microcomputer automatic cutting machine (ZQ2402) were supplied by Kinbio Tech Co., Ltd. (Shanghai, China).
The blank serum used to optimize the parameters was purchased from Sigma. The 10 negative samples were obtained from Wuhan Jinyintan Hospital and stored at −80 °C until use. The 3 positive samples were donated by the vaccinated with informed consent.