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6 protocols using alkaline phosphatase conjugated to streptavidin

1

Quantifying Antigen-Specific Antibody Responses

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Multi screen 96-well filtration plates (Millipore, Billerica, MA) were coated with 4-hydroxy-3-nitrophenyl acetyl conjugated to bovine serum albumin (NP19-BSA, Biosearch Technologies, Petaluma, CA) at a concentration of 10 μg/ml overnight at 4°C. Splenocyte suspensions from immunized mice were initially plated at 1 × 106 cells per well and then diluted serially (1:2) in RPMI1640 medium containing 10% FBS and incubated for six hours at 37°C. Bound antibodies were detected using biotinylated anti-mouse IgM and IgG Abs (Jackson Immunoresearch Laboratories, West, Grove, PA) followed by streptavidin conjugated to alkaline phosphatase (Vector Laboratories, Burlingame, CA). The plates were developed using the Vector Blue Alkaline–Phosphatase Substrate kit III (Vector Laboratories, Burlingame, CA). ELISpots were counted using a computerized imaging video system (Cellular Technology, Shaker Heights, OH).
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2

Lectin Immunosorbent Assay for Glycan Detection

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Lectin immunosorbent assay is a method akin to enzyme-linked immunosorbent assays, using lectins as probes to identify glycan structures.[24 (link)] Buffer composition was 10 mM phosphate-buffered saline (PBS) containing 150 mM sodium chloride, pH 7.2, at 4°C. The wells containing the synpatosomal lysates were washed several times with PBS containing 0.05% (w/v) Tween 20 and blocked with bovine serum albumin. A total of 100 μL of biotinylated Ulex europaeus agglutinin I lectin at 0.6 μg/mL (Vector Laboratories, Burlingame, CA) in PBS was added to each well and allowed to incubate for 1 hour. A total of 100 μL of 1:3000 dilution of 1 mg/mL streptavidin conjugated to alkaline phosphatase (Vector Laboratories) was added and signal was developed by incubation for 30 minutes with 100 μL of 1 mg/mL para-nitrophenyl phosphate substrate (Sigma-Aldrich). The reaction was stopped using 50 μL of 3 M sodium bicarbonate. Negative and positive controls were used for all of the experiments.
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3

Virus-like Particle ELISA Assay

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Virus-like particles containing protease-purified Env trimers were prepared as previously described50 (link),51 (link),91 (link), and ELISAs on these VLPs were performed as described51 (link). Briefly, Immulon II plates were coated overnight at 4°C with VLPs at 20 times their concentration in transfection supernatants. Wells were washed with PBS and then blocked with 4% bovine serum albumin/10% fetal bovine serum in PBS. Various biotinylated monoclonal antibodies (biotinylated using sulfo-NHS-Xbiotin, Thermo), and CD4-IgG2 were then titrated in the presence or absence of a fixed concentration of 2μg/ml soluble CD4. Alkaline phosphatase conjugated to streptavidin (Vector Laboratories, Burlingame, CA; to detect biotinylated mAbs) or anti-Fc (Accurate, Westbury, NY; to detect CD4-IgG2) and SigmaFAST p-nitrophenyl phosphate tablets (Sigma) were then used to detect binding. Plates were read at 405 nm.
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4

Annexin-A8 Expression in Wdr72 Mutant Mice

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Mandibles from Wdr72+/+Wdr72+/−,Wdr72−/− litermates (n = 4) were immediately immerse-fixed in 4% paraformaldehyde (PFA)/0.06 M cacodylate buffer (pH 7.3) overnight and decalcified in 8% EDTA (pH 7.2) for 2 weeks at 4 °C. Samples were then dehydrated and paraffin-processed for routine embedding and sectioning. Sagittal incisor sections at 5 μm were deparaffinized and rehydrated, followed by incubation with 10% swine serum for blocking. Rabbit polyclonal antibody to annexin-A8 (Thermo Fisher Scientific) (PA5-31479) was used at 1:500 dilution. Following overnight incubation at 25 °C, sections were washed a biotinylated secondary antibody (Dako, Carpinteria, CA) was added for 1 h at 25 °C. Alkaline phosphatase conjugated to streptavidin (Vector Laboratories Inc., Burlingame, CA) was used to visualize the colorimetric reaction. Sections were then counterstained using methyl green (Dako, Carpinteria, CA).
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5

Virus-like Particle ELISA Assay

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Virus-like particles containing protease-purified Env trimers were prepared as previously described50 (link),51 (link),91 (link), and ELISAs on these VLPs were performed as described51 (link). Briefly, Immulon II plates were coated overnight at 4°C with VLPs at 20 times their concentration in transfection supernatants. Wells were washed with PBS and then blocked with 4% bovine serum albumin/10% fetal bovine serum in PBS. Various biotinylated monoclonal antibodies (biotinylated using sulfo-NHS-Xbiotin, Thermo), and CD4-IgG2 were then titrated in the presence or absence of a fixed concentration of 2μg/ml soluble CD4. Alkaline phosphatase conjugated to streptavidin (Vector Laboratories, Burlingame, CA; to detect biotinylated mAbs) or anti-Fc (Accurate, Westbury, NY; to detect CD4-IgG2) and SigmaFAST p-nitrophenyl phosphate tablets (Sigma) were then used to detect binding. Plates were read at 405 nm.
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6

Detailed Protocol for In-Vitro Cytotoxicity Assays

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Dulbecco’s Modified Eagle Medium (DMEM), Amplex UltraRed, and 4-methylumbelliferyl phosphate (4-MUP) were obtained from Invitrogen (Carlsbad, CA, USA). The CYQUANT MTT Cell Viability Assay, which is based on the cellular conversion of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to an insoluble formazan product, Hoechst (H33342) dye, and Nunc Lab Tek 8-well chamber slides were from Thermo Fisher (Cambridge, MA USA). Alkaline Phosphatase Conjugated to Streptavidin was purchased from Vector Laboratories (Burlingame, CA, USA). Details pertaining to the Agent Orange chemicals are listed in Table 1, and antibodies used in immunoassays are listed in Table 2. All other fine chemicals were purchased from Thermo Fisher (Cambridge, MA USA), Sigma-Aldrich (St. Louis, MO, USA), or CalBiochem (Carlsbad, CA, USA).
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