Immulon 4hbx

Ninety-six-well, flat-bottomed microtiter plates (Immulon 4HBX, Thermo Fisher Scientific, Waltham, MA) were coated with either 5 µg/ml antigen peptide in DPBS or DPBS only overnight at 4°C. Plates were washed 3 times with 0.1% Tween 20-DPBS (T-DPBS) and then blocked with 1% BSA in DPBS for 2 hr at room temperature. Plates were washed 5 times with T-DPBS and incubated with the rabbit anti-PP antibody, whole antisera (without antigen-affinity purification), or flow-through solution in triplicate in a serial dilution for 90 min at room temperature. After washing, the wells were incubated with goat anti-rabbit IgG antibodies conjugated with HRP in DPBS (1∶3000) for 1 hr at room temperature. They were washed and detected with 3, 3, 5, 5′-Tetramethyl Benzidine substrate solution (TMB, Pierce, Rockford, IL) for 30 min at room temperature. After the addition of 2 N HCl to stop the colorimetric reaction, optical density was measured at 450 nm using a microtiter plate reader (Multiskan, Thermo Scientific Japan, Tokyo, Japan).

ELISA assays were performed as previously described [10 (link)]. Briefly, Immulon^® 4HBX (Thermo Fisher Scientific, Waltham, MA, USA) or Costar EIA/RIA (Corning, Corning, NY, USA) plates were coated with 100 ng/well of recombinant SARS-CoV-2 RBD protein (based on USA/WA1/2020 strain), incubated with heat inactivated serum samples at a starting dilution of 1:50, and then further serially diluted 3-fold [20 (link)]. IgG antibodies were detected using horseradish peroxidase (HRP)-conjugated goat antihuman IgG detection antibody (Southern Biotech, Birmingham, AL, USA) at a 1:4000 dilution, and colorimetric development was accomplished using 100 μL of 0.1% 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Bioworld, Dublin, OH, USA) solution with 0.05% H₂O₂ for 18 min at 37 °C. The reaction was terminated with 50 μL of 1% (w/v) SDS (VWR International, Radnor, PA, USA). Colorimetric absorbance was measured at 414 nm using a PowerWaveXS plate reader (Biotek, Winooski, VT, USA). All samples and controls were run in duplicate, and the mean of the two blank-adjusted optical density (OD) values were used in downstream analyses. IgG equivalent concentrations were calculated based on a 7-point standard curve generated by a human IgG reference protein from plasma (Athens Research and Technology, Athens, GA, USA), and verified on each plate using human sera of known concentrations.

After transfection for 48 h, cells (2 × 10⁴/well) were plated in every well of 96-well plates, and 10 μL CCK-8 solution (CK04, Dojindo, Tokyo, Japan) was added to each well at 24 h, 48 h and 72 h. Next, the cells were incubated for 1 h. The absorbance was detected at a wavelength of 450 nm using a microtiter plate (Immulon 4HBX, Thermo Labsystems, Rochester, NY) [17 (link)].

Flat-bottom 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were coated with 2 µg/ml protein (50 µl/well) diluted in coating solution (KPL) overnight at 4°C. The following day, plates were washed three times with PBS containing 0.1% Tween 20 (Fisher Scientific) (T-PBS) and were blocked with 220 µl of blocking solution (3% goat serum [Gibco] and 0.5% milk powder in T-PBS) for 1 h at room temperature. After removal of the blocking buffer, serum samples at a 1:100 starting concentration were 2-fold serially diluted in blocking solution. After 2 h of incubation at room temperature, the plates were washed three times with T-PBS. Anti-human IgG (Fab specific) horseradish peroxidase (HRP)-conjugated antibody (Sigma-Aldrich) diluted 1:3,000 in blocking solution was added to all wells (50 µl/well). After a 1-h incubation at room temperature, the plates were washed four times with T-PBS and developed with 100 µl/well of SigmaFast ο-phenylenediamine dihydrochloride (OPD; Sigma) for 10 min. The reaction was stopped with 50 µl of 3 M hydrochloric acid (HCl) and then subsequently read at an absorbance of 490 nm with a Synergy H1 hybrid multimode microplate reader (BioTek). The average background plus three standard deviations were calculated and used as the lower limit for area under the curve (AUC) analysis using GraphPad Prism software.

Soluble TREM‐Fc family member proteins were purified and eluted as previously described for the BioLayer Interferometry. Purified samples were coated overnight at 4°C in 96‐well plates (Immulon 4HBX, Thermo Scientific, NY, USA) in 100 mM NaCO₃ pH 11. Plates washed with PBS twice and then blocked 3 h at room temperature with blocking buffer (Block Ace, Abd Serotec, Japan). Blocked samples were incubated with Aβ42 monomers, Aβ42 oligomers, or Aβ42 fibrils at specified concentration for 1 h at room temperature. Aβ was detected with 4G8‐HRP, and soluble TREM‐Fc samples were detected with anti‐human IgG HRP to insure similar coating level of proteins. Samples were developed with TMB substrate (KPL, Maryland, USA), and the reaction was stopped by adding 6.67% O‐phosphoric acid (85%). The signal produced by HRP and TMB substrate was quantified by measuring the absorbance at 450 nM.