Vaccine and immunoglobulin ELISPOTs were performed as previously described46 (link). In short, ELISPOT filter plates were coated overnight with influenza vaccine, anti-IgG or anti-IgA at 4C. Plates were sorted and blocked with RPMI with 10% FCS for 2 hours at 37C. Sorted cells in RPMI with 10% FCS, 1% HEPES, 1% L-Glutamine, and 1% penicillin/streptomycin were added to the plates in a 1:2 dilution series. Cells were incubated for 5 hours or overnight. Plates were then washed with PBS with .05% Tween and biotinylated anti-IgG or anti-IgA (Southern Biotech) was added at a 1:1000 dilution. Streptavidin-AP (Southern Biotech) was used as a secondary antibody and the ELISPOT was revealed using NBT/BCIP (Thermo Scientific).
Streptavidin ap
Manufactured by Southern Biotech
Sourced in United States
Streptavidin-AP is a conjugate of streptavidin and alkaline phosphatase. Streptavidin is a tetrameric protein that binds strongly to the vitamin biotin. Alkaline phosphatase is an enzyme that catalyzes the hydrolysis of phosphate esters. The streptavidin-AP conjugate can be used to detect and quantify biotinylated molecules in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphate substrate, by Merck Group (2 mentions)
Baf413, by R&D Systems (2 mentions)
Ready set go ifnγ elisa kit, by Thermo Fisher Scientific (2 mentions)
Np20 bsa, by Biosearch Technologies (2 mentions)
Biotinylated anti igg or anti iga, by Southern Biotech (1 mentions)
Nbt bcip, by Thermo Fisher Scientific (1 mentions)
Elisa kits for ifn γ, by Thermo Fisher Scientific (1 mentions)
Ige biotin, by BioLegend (1 mentions)
Multiscreen filter plate, by Merck Group (1 mentions)
Biotinylated anti igm, by Southern Biotech (1 mentions)
Eos 20d digital camera, by Canon (1 mentions)
Sigmafast bcip nbt, by Merck Group (1 mentions)
Nbt bcip substrate solution, by Thermo Fisher Scientific (1 mentions)
Msips4w10, by Merck Group (1 mentions)
Mt78 145, by Mabtech (1 mentions)
Ctl immunospot analyzer, by Cellular Technology (1 mentions)
Alkaline buffer, by Merck Group (1 mentions)
Eia ria 96 well plate, by Corning (1 mentions)
Elispot plate, by Merck Group (1 mentions)
11 protocols using streptavidin ap
1
Influenza Vaccine ELISPOT Assay
2
Cytokine and IgE ELISA Protocols
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Ready-SET-go! ELISA kits for IFN-γ, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17a and TGF-β1 (all from eBioscience) were used according to manufacturer’s instructions. IgE ELISA reagents (kind gift of Nicola Harris, EPFL), including IgE standard grown from TIB-141 (ATCC, Manassas, VA, USA), IgE unlabeled coating antibody grown from 6HD5, IgE biotin (Biolegend) and streptavidin-AP (Southern Biotech, Birmingham, AL, USA) were used as previously described34 (link).
3
Antibody and Cytokine Quantification in Murine Infection
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Serum was collected at different time points after infection. The following in house ELISA antibodies were used in IgE, IgG1, and IgM ELISAs47 (link): anti-IgE capture, biotinylated anti-mouse IgE, anti- mouse IgG1 capture, anti-mouse IgG1 alkaline phosphatase (AP), anti-mouse IgM capture, and anti-mouse IgM AP. The 96-well plate was coated with the capture antibody; serially diluted samples were added and followed by detection antibody. IgE, IgG1, and IgM standards were used to generate a standard curve. Plates were developed by adding alkaline phosphate substrate (Sigma-Aldrich) and read by a spectrophotometer at 405 and 650nm.
Cell culture supernatants were examined for IL-13 and interferon (IFN) cytokines. For IL-13, 96-well plates were coated with anti-mouse IL-13 capture antibody (6μg/ml, #MAF413, R&D Systems, Minneapolis, MN) for 1h at 37°C. Plates were blocked for 1h at 37°C with 10% fetal bovine serum in PBS. Samples were added to plates neat and diluted 1:10 for 2hr at 37°C. Recombinant IL-13 was used to generate a standard curve. Biotinylated anti-mouse IL-13 detected antibody (0.2μg/ml, #BAF413, R&D Systems) was added for 1h at 37°C, followed by Streptavidin-AP (Southern Biotech) for 1h at 37°C. Plate was developed as described above. IFNγ was detected in the supernatants using Ready-Set-Go! IFNγ ELISA kit (eBioscience, San Diego, CA) per manufacturer's instructions.
Cell culture supernatants were examined for IL-13 and interferon (IFN) cytokines. For IL-13, 96-well plates were coated with anti-mouse IL-13 capture antibody (6μg/ml, #MAF413, R&D Systems, Minneapolis, MN) for 1h at 37°C. Plates were blocked for 1h at 37°C with 10% fetal bovine serum in PBS. Samples were added to plates neat and diluted 1:10 for 2hr at 37°C. Recombinant IL-13 was used to generate a standard curve. Biotinylated anti-mouse IL-13 detected antibody (0.2μg/ml, #BAF413, R&D Systems) was added for 1h at 37°C, followed by Streptavidin-AP (Southern Biotech) for 1h at 37°C. Plate was developed as described above. IFNγ was detected in the supernatants using Ready-Set-Go! IFNγ ELISA kit (eBioscience, San Diego, CA) per manufacturer's instructions.
4
ELISPOT Assay for IgG1-Secreting Cells
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One million splenocytes were seeded in each well of the MultiScreen filter plate (Milipore, Darmstadt, Germany) coated with NP20-BSA (Biosearch Technologies, Novato, CA, USA) and blocked with BSA for incubation. After washing, the plate was incubated with biotin-conjugated anti-IgG1 antibodies (1/1000, SouthernBiotech, Birmingham, AL, USA) for 1 h at room temperature, and with streptavidin-AP (1/1000, SouthernBiotech, Birmingham, AL, USA) for another 0.5 h. Then, the antibody-secreting cells were visualized using substrates (Mabtech, Cincinnati, OH, USA).
5
ELISPOT Assay for Antibody-Secreting Cells
6
NP-BSA ELISPOT Assay for Antibody-Secreting Cells
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ELISPOT assay was performed as described previously55 (link). In brief, MultiScreen filter plates with 0.45 μm PVDF membrane (Milipore) were coated with 1 μg per well NP20-BSA (Biosearch Technologies) in PBS overnight at 4°C and blocked with 2% BSA in PBS for 2 h at room temperature. One million splenocytes or three million BM cells were seeded in each well, and then the plate was incubated at 37°C for 2 h. After washing, the plate was incubated with Biotinylated anti-IgM (1: 1000, SouthernBiotech, 1140-08) or anti-IgG1 (1: 1000, SouthernBiotech, 1144-08) antibodies for 1 h at room temperature, followed by streptavidin-AP (1:1000, SouthernBiotech, 7105-04) incubation for 0.5 h at room temperature. The substrates (MABTECH) were added to each well for visualization of antibody-secreting cells.
7
MPER-specific Antibody-Forming Cell ELISpot Assay
8
Quantifying Antigen-Specific Antibody Secreting Cells
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Multiscreen 96-well filter PVDF membrane plates (EMD Millipore, MSIPS4W10) were coated with either pertussis antigen pool (5 μg/mL of PT, FHA, Fim 2/3, and PRN) or 109 CFU/mL heat-killed Bordetella pertussis in 100 mL DPBS overnight. Cryopreserved mononuclear bone marrow cells were thawed, counted, and assessed for viability. Next, 4 × 105 viable mononuclear bone marrow cells were added to each well and incubated in a 37 °C incubator with 5% CO2. Twenty hours post incubation, culture media was removed, and sterile water was added to each well to lyse the cells. 1 µg/mL biotinylated anti-human IgG mAb MT78/145 (Mabtech, 3850-6) was added followed by Streptavidin-AP (Southern Biotechnology, 7100-04) and BCIP/NBT substrate solution (Thermo Scientific, 34042) in order to visualize the spots. The development reaction was stopped by rinsing the wells with tap water. The plates were air dried overnight in the dark at room temperature and a CTL ImmunoSpot Analyzer (Cellular Technology Limited) was used to scan, count and QC the wells in each plate. Results for each baboon were expressed as the numbers of antibody secreting long-lived plasma cells per 106 mononuclear bone marrow cells.
9
Quantification of Human Antibody-Secreting Cells
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EIA/RIA 96-well plates (Corning) were coated with goat anti–human κ or λ Abs at 10 µg/ml (2060-01, 2070-01; SouthernBiotech) in PBS and left overnight at 4°C, washed three times with PBS, and blocked with PBS/1% gelatin for 2 h at RT. 10 × 106 spleen cells from hu-mice were resuspended in 400 µl RPMI media supplemented with 10% FBS, GlutaMAX, pen/strep, β-ME, and 10% protein G–absorbed FBS. 400-µl cell suspensions were added in the first well of each plate followed by twofold serial dilutions, and plates were incubated overnight at 37°C. Plates were washed four times in PBS/0.1% Tween followed by the addition of biotinylated goat anti–human Ig(H+L) Abs (2010-08; SouthernBiotech) at 1 µg/ml in staining buffer. Plates were incubated for 2 h at RT in the dark and washed three times with PBS/0.1% Tween, followed by incubation with a 1:2,000 dilution of Streptavidin-AP (SouthernBiotech) in staining buffer for 1.5 h in the dark at RT. Plates were washed four times with PBS/0.1% Tween, and 100 µl of developing buffer (1M alkaline buffer [Sigma-Aldrich], 5 mM MgCl2, 1 mM Triton X-405, 0.1% NaN3, and BCID [1% wt/vol; Thermo Fisher Scientific]) was added per well, left to incubate overnight at 4°C, washed the next day with diH2O three times, and allowed to dry before scanning. Spots were counted manually.
10
Quantification of Cytokine Secretion
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IL-4 and IFN-γ produced by splenocytes were examined by using ELISPOT at the end of study. Spleens were processed to make single cell solution after red blood cell lysis. 5 × 105 cells per well were added to anti-mouse IL-4 or IFN-γ antibody (Invitrogen, Carlsbad, CA) coated PVDF membrane ELISPOT plates (EMD Millipore, Billerica, MA). Cells were cultured with or without 1 μg/mL CryJ2 protein. Cells incubated with ConA were used as a positive control. 48 hours later, plates were washed and then incubated with biotin-conjugated IL-4 or IFN-γ detection antibody followed by Streptavidin-AP (Southern Biotech, Birmingham, AL). Plates were developed with BCIP substrate (KPL) and spots were counted with Advanced Imaging Devices GmbH system (Strassberg, Germany).
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