Nunc maxisorp

96-well plates (Nunc MaxiSorp™, Invitrogen™) were coated with 200 ng/well of recombinant CD166 protein (sumo-tagged human CD166 ECD protein was produced at CytomX Therapeutics, Inc.) in 0.05 M carbonate buffer. Plates were washed with 3 × 300 µL Tris-buffered saline (TBS), 0.1% Tween 20 (wash buffer), followed by blocking with TBS + 0.5% casein (block) for 1 h at room temperature. Plates were washed three times and incubated with 80 µL of the indicated concentrations of CX-090 or CX-191 for 1 h at room temperature. Plates were washed and incubated with 80 µL of detection antibody (AffiniPure Anti-human IgG, Jackson ImmunoResearch cat #109-035-088) at 1 to 10,000 dilution for 30 to 45 min at room temperature. Detection was performed by the addition of 3,3′,5,5′-tetramethylbenzidine substrate (1-Step Ultra-TMB, Pierce) followed by an equal volume of 1 M hydrochloric acid. Absorbance at 450 nm was then measured and reported as optical density (OD) 450 nm. Data were analyzed using Prism Graphpad, and apparent equilibrium binding constants (K_app) were determined using nonlinear regression four parameter logistic (4-PL) analysis.

ViPS-specific IgM and IgG in the blood were measured by coating 96-well microtiter plates (Nunc MaxiSorp; Invitrogen, Carlsbad, CA) with 2 μg/ml ViPS purified from S. Typhi clinical isolate C6524 (47 (link)) in DPBS overnight at room temperature. All plates were washed and blocked with 1% BSA in PBS (pH 7.2) (blocking buffer) for 1 h at room temperature. Blood samples from immunized mice were diluted to 1:25 for IgG detection and 1:50 for IgM detection in blocking buffer, and samples were centrifuged (800 × g for 10 min) and cell-free supernatant was used. The dilutions, 1:25 for IgG and 1:50 for IgM, were chosen after evaluating various serum dilutions within the linear range by ELISA. Bound IgM or IgG was measured using HRP-conjugated goat anti-mouse IgM or IgG (Bethyl Laboratories, Montgomery, TX). Because ViPS-specific mouse IgM and IgG reference standards are not available, and the ViPS-specific Abs in mice are likely to be of oligoclonal nature with varying affinities, the Ag-specific Ab levels in the current study were interpreted as nanogram per microliter “equivalents” using normal mouse serum IgM or IgG standards (Bethyl Laboratories, Montgomery, TX).

For quantitative analysis of 4-HNE adducts in serum, an enzyme-linked immunosorbent assay (ELISA) established by our group was used (Monroe and Anderson, 2019 (link)). In brief, standards of 4-HNE-protein adducts were first made by incubating serial dilutions of 4-HNE (Cayman chemicals, United States) in 1 mg/mL BSA/50 mM sodium phosphate buffer (pH = 7.4) at 37 C° for 24 h. Serum and standards were loaded in duplicate on immunolon-coated 96-well plates (Nunc MaxiSorp, Invitrogen, United States) and incubated overnight at 4C° with a continuous rocking. The plate was then blocked with 10% BSA for 2 h followed by overnight incubation with 4-HNE polyclonal primary antibody (Sigma-Aldrich). Next the plate was incubated with a HRP-conjugated mouse anti-goat secondary antibody followed by incubation with 55 µM Amplex red (fluorogenic reagent) for 30 min at room temperature. Fluorescence intensity of each well was then measured using the BioTek Synergy microplate reader at 530/595 nm (ex/em), concentrations of 4-HNE protein adducts in the serum samples were determined against the standard curve.

All ELISAs were performed on Nunc Maxisorp 96-wells microtiter plates (Invitrogen), each step in a final volume of 100 µL at RT. Between incubation steps, plates were washed five times with PBS 0.02% (w/v) Tween-20, using a Biotek 405 LSRS (Biotek Instruments, Winooski, VT, USA). Assays were developed by addition of 100 µg/mL 3,5,3′,5′-Tetramethylbenzidine in 0.11 M sodium acetate containing 0.003% (v/v) H₂O₂, pH 5.5, and stopped by addition of 100 µL 2 M H₂SO₄. Absorbance was measured at 450 nm and corrected for absorbance at 540 nm using a Synergy 2 Multi-Mode plate reader (BioTek Instruments).

Antigen-specific total IgG titers were measured by ELISA. 96-well plates (Nunc MaxiSorp, Invitrogen) were coated overnight at 4 °C with 0.1 µg/well Tag-muIL-1β in PBS, pH 7.4. Plates were blocked with 0.5% skimmed milk in PBS O/N at 4 °C. Mouse serum was diluted in a 3-fold dilution starting from 1:200, followed by incubation for 1 h at RT. Plates were washed 3 times in PBS in between steps. Total serum IgG was detected using Horseradish peroxidase (HRP) conjugated goat-anti mouse IgG (Invitrogen)diluted 1:1000 in blocking buffer and incubated for 1 h at RT. Plates were developed with TMB X-tra substrate (Kem-En-Tec, Taastrup, Denmark) and the absorbance was measured at 450 nm. For the avidity assay, plates were run in duplicates, followed by a 5 min PBS or 8M urea wash, before incubation with the secondary antibody.

To measure antigen‐specific antibody responses, enzyme‐linked immunosorbent assay (ELISA) Nunc MaxiSorp flat‐bottom plates (Invitrogen 44‐2404‐21) were coated with 2.5 µg mL⁻¹ with the mixture of four antigens in phosphate coating buffer (0.1 m Na2HPO4 in deionized water, pH 9.0) overnight at 4 °C. Next day, plates were blocked with 1% bovine serum albumin in phosphate buffered saline‐Tween (1X PBS, 0.05% Tween) for 2h at room temperature. Plates were then washed 4x with PBS‐T and incubated overnight at 4 °C with serially diluted sera collected from mice in PBS‐T. The next day, wells were washed 4x with PBS‐T and incubated for 1h at room temperature either with HRP‐IgG1 (SouthernBiotech 1070‐05) (1:4,000) or HRP‐IgG2a (SouthernBiotech 1080‐05) (1:4000). Wells were subsequently washed 4x with PBS‐T and developed with 2,2‐azinobis(3‐ethylbenzthiazolinesulfonic acid) (ABTS) (VWR 95059‐146) substrate for 5 min at room temperature. At the end of the incubation, the reaction was stopped with 10% SDS solution. Absorbance was measured at 405 nm to determine endpoint antibody titers.

An ELISA was used to assess the vaccine-induced immune responses. The 96-well microtiter plates (Nunc MaxiSorp, Invitrogen, Waltham, MA, USA) were coated with 0.1 μg/well recombinant HA_stem.Tag (produced in baculovirus expression system) or SARS-CoV-2 Spike protein aa35-1208 (produced in ExpreS2, ExpreS2ion Biotechnologies, Hørsholm, Denmark) and incubated overnight at 4 °C. Plates were blocked with 0.5% skimmed milk in PBS for 1 h at RT with shaking. Mouse sera were diluted in PBS + 0.5% skimmed milk in a 3-fold serial dilution, starting at a 1 in 50 dilution. A total of 50 μL of the diluted sera was then transferred to the plates and incubated for 1 h at RT with shaking. Plates were washed 3 times with PBS and probed with a goat anti-mouse HRP conjugated secondary antibody (anti-IgG (Life technologies, A16072); IgG1 (Invitrogen, A10551); IgG2a (Invitrogen, M32207); IgG2b (Invitrogen, M32407); or IgG3 (Thermo Fisher, M32707)) for 1 h at RT with shaking. All secondary antibodies were used at a dilution of 1:1000 in PBS + 0.5% skimmed milk. Plates were washed 3 times with PBS and developed with TMB X-tra substrate (Kem-En-Tec, Taastrup, Denmark). Absorbance at 450 nm was measured on a HiPo MPP-96 microplate reader (BioSan, Riga, Latvia).