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Streptavidin alp

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Streptavidin-ALP is a conjugate of streptavidin, a protein with a high affinity for biotin, and alkaline phosphatase (ALP), an enzyme that catalyzes the hydrolysis of phosphate esters. This conjugate is used as a detection reagent in various bioanalytical techniques, such as enzyme-linked immunosorbent assays (ELISAs) and western blotting, where it can be used to amplify and visualize the signal from biotin-labeled targets.

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Lab products found in correlation

Bcip nbt plus substrate, by Mabtech (4 mentions) Color development buffer, by Bio-Rad (3 mentions) Tween 20, by Thermo Fisher Scientific (3 mentions) Anti cd28 clone cd28, by BioLegend (3 mentions) Immobilon p 96 well microtiter plates, by Merck Group (3 mentions) Anti ifn γ clone dk1, by Mabtech (3 mentions) Anti ifn γ clone 1 dk1, by Mabtech (3 mentions) Anti ifn γ clone b6 1, by Mabtech (3 mentions) Ap conjugated substrate, by Bio-Rad (3 mentions) Anti cd3 clone okt3, by BioLegend (3 mentions) Nbt bcip substrate, by Thermo Fisher Scientific (3 mentions) Multiscreen ip 96 well plates, by Merck Group (3 mentions) Goat anti mouse ig, by Southern Biotech (2 mentions) Biotinylated anti mouse igg, by Merck Group (2 mentions) Bcip nbt plus, by Mabtech (2 mentions) Clone 7 b6 1, by Mabtech (2 mentions) Nbt bcip, by Thermo Fisher Scientific (2 mentions) Bcip nbt, by Mabtech (2 mentions) Anti human igg, by Mabtech (1 mentions) Multiscreen ip plate, by Merck Group (1 mentions)

28 protocols using streptavidin alp

1

Quantification of Antibody-Secreting Cells

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Milipore multiscreen-IP plates (Milipore, MA) were prepared per manufacturer’s instruction and coated with appropriate antibodies in PBS: 2ug/mL Goat Anti-Mouse Ig (Southern Biotech), or 4–8ug/mL anti-human IgG (Rockland Biosciences, NY). The following day, plates were washed and blocked with complete medium. Bulk cell suspensions were prepared, diluted appropriately and incubated in complete IMDM (10% FCS, 1.0% Pen/Strep) for 18 hours at 37°C and 5% CO2. Standard ELISPOT procedures were followed using biotinylated anti-mouse IgG (Sigma) or anti-human IgG (mABtech) followed by streptavidin-ALP (mABTech). Assay was developed with NBT/BCIP Substrate (ThermoScientific) and analyzed using CTL Immunospot 5.0 software (Cellular Tech Ltd). Data were calculated as antibody-secreting (ASC) spots/106 lymphocytes.
Tedesco D., Thapa M., Gumber S., Elrod E.J., Rahman K., Ibegbu C.C., Magliocca J.F., Adams A.B., Anania F, & Grakoui A. (2016). CD4+ Foxp3+ T cells promote aberrant IgG production and maintain CD8+ T cell suppression during chronic liver disease. Hepatology (Baltimore, Md.), 65(2), 661-677.
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2

Granzyme B ELISPOT Assay for HIV Antigens

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Mabtech Granzyme B (clone GB10, catalog 3486-2 A) ELISPOT assays against whole gene product peptides with overlap 15-mer coverage of HIV-Gag (catalog ARP-12425), HIV-Env (catalog ARP-12540), HIV-Pol (catalog ARP-12438), and HIV-Nef (catalog ARP-12545) from the NIH HIV Reagent Program and CMVpp65 peptide pool (catalog PM-PP65-2) were performed in triplicate. Multiscreen IP 96-well plates (MilliporeSigma) were coated with 7.5 μg/mL of anti–Granzyme B antibody (clone GB10, Mabtech catalog 3486-2 A) in sterile water and incubated overnight. Plates were washed, and PBMCs were added at 100,000–200,000 cells/well and stimulated with peptide pools and 0.5% DMSO and phytohemagglutinin at 2 μg/mL as negative and positive controls, respectively. Plates were incubated overnight; washed and biotinylated antibody was added (anti–Granzyme B antibody clone MT8610 from Mabtech) and incubated for 2 hours. Plates were developed with Streptavidin-ALP from Mabtech and with Color Development Buffer (Bio-Rad). Plates were washed and dried overnight and spots were counted. Responses against whole gene product peptide pools were background subtracted (thus, nonzero responses were more than 1 time background), but no other ad hoc empirical cutoff was applied — consistent with other studies examining correlations with objectively reported T cell responses as assessed by ELISPOT assay.
Copertino DC J.r., Holmberg C.S., Weiler J., Ward A.R., Howard J.N., Levinger C., Pang A.P., Corley M.J., Dündar F., Zumbo P., Betel D., Gandhi R.T., McMahon D.K., Bosch R.J., Linden N., Macatangay B.J., Cyktor J.C., Eron J.J., Mellors J.W., Kovacs C., Benko E., Bosque A, & Jones R.B. (2023). The latency-reversing agent HODHBt synergizes with IL-15 to enhance cytotoxic function of HIV-specific T cells. JCI Insight, 8(18), e169028.
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3

IFN-γ ELISPOT Assay for T Cell Responses

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These experiments were also performed as described in a previous publication13 (link). IFN-γ ELISPOT assays were performed using 96-well MultiScreen Filter Plates (Millipore), coated with 2 μg/mL anti-human IFN-γ mAb overnight (1-D1K, Mabtech). Plates were washed with PBS and blocked with complete RPMI before use. 5 × 103 T cells were co-cultured with 1 × 104 autologous CD4+ and CD8+ T cell-depleted PBMCs (APC). Peptides (10 μg/mL) were directly added to the ELISPOT wells with APCs and incubated with T cells overnight in complete RPMI at 37 °C. The plates were rinsed with PBS containing 0.05% Tween-20 and then 1 μg/mL anti-human IFN-γ mAb (7-B6-1-Biotin, Mabtech) was added, followed by Streptavidin-ALP (Mabtech). After rinsing, SIGMA FAST BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; Sigma-Aldrich) was used to develop the immunospots, then the spots were imaged and counted (Cellular Technology Limited).
Truex N.L., Holden R.L., Wang B.Y., Chen P.G., Hanna S., Hu Z., Shetty K., Olive O., Neuberg D., Hacohen N., Keskin D.B., Ott P.A., Wu C.J, & Pentelute B.L. (2020). Automated Flow Synthesis of Tumor Neoantigen Peptides for Personalized Immunotherapy. Scientific Reports, 10, 723.
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4

ELISPOT Quantification of IgG and IgA

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Briefly, ELISPOT plates (MSIPSW10, Millipore Sigma) were activated with 70% EtOH before being washed 4x with sterile dH2O. Plates were coated overnight with purified anti-human IgG (MT91/145, Mabtech), anti-monkey IgA (polyclonal, Rockland), or anti-human IgA (MT57, Mabtech). Cells to be assayed were plated in duplicate and serially diluted down the ELISPOT plate before overnight incubation at 37 °C in a 5% CO2 incubator. The following day, cells were lysed by incubation with dH2O before being washed 4× with PBS-T. Secreted IgG or IgA were detected using biotinylated anti-human IgG (MT78/145, Mabtech) or biotinylated anti-human IgA (MT57, Mabtech) followed by incubation with streptavidin-ALP (Mabtech). ELISPOT plates were developed with NBT/BCIP (ThermoFisher Scientific) before being quenched with dH2O. All washes between incubation steps were performed using PBS-T. Plates were read and quantified on an Immunospot S6 (Cellular Technology Limited (CTL)) using CTL Immunocapture (v7.0.16.1, CTL) and CTL Biospot (v7.0.3.4, CTL). Manual inspection of automated counting results was performed as quality control during analysis.
Staupe R.P., Lodge K.E., Thambi N., Toole D., Tamburino A.M., Chang D., Howell B.J., Hazuda D.J., Vora K.A, & Sullivan N.L. (2022). Single cell multi-omic reference atlases of non-human primate immune tissues reveals CD102 as a biomarker for long-lived plasma cells. Communications Biology, 5, 1399.
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5

Quantifying T-cell responses to tumor antigens

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1 x 104 stimulated T cells were incubated with peptide-loaded dendritic cells (DCs) at 1:30 ratio on a 96 well ELISpot plates pre-coated with anti-human IFNγ antibody (mAb 1-DIK, Mabtech, UK) overnight at 37°C. DC were differentiated from autologous CD14+ monocytes, according to Miltenyi manufacturer’s protocol in ImmunoCult™ DC differentiation media (Stemcell Technologies, UK) for two weeks. DCs were loaded with 1 µg/ml CEFT, MAGED4B or FJX1 OPP. Samples were plated in duplicate or triplicate. Spots were detected with a biotin-conjugated human IFNγ antibody (mAb 7-B6-1, Mabtech UK) followed by incubation with Streptavidin ALP (Mabtech, UK) and BCIP/NBT (Thermo, UK). Plates were scanned using ImmunoSpots reader (AID, UK). The mean values were represented as spot-forming cells (SFCs) per 1 x 104 T cells. Levels were considered positive if at least two times above medium control.
Wang C., Zainal N.S., Chai S.J., Dickie J., Gan C.P., Zulaziz N., Lye B.K., Sutavani R.V., Ottensmeier C.H., King E.V., Abraham M.T., Ismail S.M., Lau S.H., Kallarakkal T.G., Mun K.S., Zain R.B., Abdul Rahman Z.A., Thomas G.J., Cheong S.C., Savelyeva N, & Lim K.P. (2021). DNA Vaccines Targeting Novel Cancer-Associated Antigens Frequently Expressed in Head and Neck Cancer Enhance the Efficacy of Checkpoint Inhibitor. Frontiers in Immunology, 12, 763086.
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6

ELISpot Assay for IgG Antibody Secreting Cells

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ELISpot for total IgG antibody secreting cells was performed as described previously (38 (link)). In brief, Milipore multiscreen plates (Milipore, MA) were coated with 2 μg/mL Goat Anti-mouse Ig (Southern Biotech). The following day, plates were washed and blocked with complete medium. CD19 magnetic bead (Miltenyi) enriched B cells (50,000 cells) were added to the plate and incubated for 18 hours at 370C and 5% CO2. Standard procedures using biotinylated anti-mouse IgG (Sigma) was followed with streptavidin-ALP (mABTech), and assay was developed using NBT/BCIP substrate (Thermo-Scientific), and analyzed using CTL Immunospot 5.0 software (Cellular Tech Ltd.). Data are represented as IgG antibody secreting cells (ASC) spots per 106 cells (IgG ASC/106 cells).
Thapa M., Tedesco D., Gumber S., Elrod E.J., Han J.H., Kitchen W.H., Magliocca J.F., Adams A.B, & Grakoui A. (2020). Blockade of B-cell activating factor (BAFF) reshapes the hepatic B cell receptor repertoire and attenuates autoantibody production in cholestatic liver disease. Journal of immunology (Baltimore, Md. : 1950), 204(12), 3117-3128.
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7

Quantifying Cellular Immune Responses

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Ethanol-activated MultiScreenHTS IP filer plates (EMDMillipore) were coated with either 100 μL of filtered (with 0.45 μM filters) Salmon Sperm DNA (5 μg/mL g/mL; ThermoFisher) or NP-OVA (10 μg/mL, Biosearchtech) overnight at 4°C. The plates were then washed with PBS and blocked for 2 hours with blocking buffer (5% FCS, 3% BSA in PBS) before they were re-washed and let dry. 50 μL of splenocytes in clone media (started with 5 x 105 cells per well with 1/3 serial dilutions) were plated and 50 μL of ACD40 (10 μg/mL) was added per well (final concentration of 5 μg/mL). The cells were incubated at 37°C for 24 hours. After that, cells and unbound cytokines were washed by incubating with PBS Tween-20 buffer for 10 minutes and then thoroughly re-washed. 50 μL of biotin-conjugated goat anti-mouse Ig (1:350 dilution in clone media, SouthernBiotech) was then added and incubated overnight at 37°C for primary staining. After rewashing, secondary staining was done for 1 hour using 50 μL of streptavidin-ALP (1:1000 dilution in 1% BSA in PBS, Mabtech). The plates were then washed and developed using 50 μL BCIP/NBT-plus substrate (Mabtech). When spots are clearly visible under a dissecting microscope, stop the development by discarding the substrate and rinse plates with tap water thoroughly. Spots were counted manually with a dissecting microscope.
Sungnak W., Wagner A., Kowalczyk M.S., Bod L., Kye Y.C., Sage P.T., Sharpe A.H., Sobel R.A., Quintana F.J., Rozenblatt-Rosen O., Regev A., Wang C., Yosef N, & Kuchroo V.K. (2020). TFR-derived Fibrinogen-like Protein 2 regulates production of autoantibodies and induction of systemic autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 205(12), 3247-3262.
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8

Quantifying IgG-secreting B Cells

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For detection of total IgG secreting B cells, plates (Millipore, Cat: MAIPSWU10) were coated with anti-mouse IgG F(ab)’ specific antibody (Jackson Immunoresearch, Cat: 115-005-072) overnight at 4 °C, then washed with PBS, and blocked with DMEM (10% FBS) 2 hours at 37°C. Cells were plated and cultured overnight, then washed and alkaline phosphatase conjugated anti-mouse IgG Fc specific antibody was added (Jackson Immunoresearch, Cat: 115-055-071). After 1 hour at room temperature, plates were washed and developed with substrate BCIP/NPT (Sigma, Cat: B5655-25tab). For detecting H2-Kd specific IgG secreting plasma cells, plates were coated with anti-mouse IgG Fc-gamma specific antibody overnight at 4°C, washed and blocked. Cells (103, 104, and 105) were then plated and cultured overnight. Plates were washed and H2-Kd-biotin monomers were added. After one hour at room temperature, plates were washed and streptavidin-ALP (Mabtech, Cat:3310-10) was added. After one additional hour incubation at room temperature, plates were then washed and developed. The numbers of spots per well were enumerated using the ImmunoSpot Analyzer (CTL Analyzers LLC).
Miller M.L., Chen J., Daniels M.D., McKeague M.G., Wang Y., Yin D., Vu V., Chong A.S, & Alegre M.L. (2016). Adoptive transfer of tracer alloreactive CD4+ TCR-transgenic T cells alters the endogenous immune response to an allograft. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(10), 2842-2853.
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9

IFNγ ELISpot assay for antigen-specific T cells

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Spleens and lymph nodes were processed through 70 µm cell strainers to obtain a single cell suspension, followed by a red blood cell lysis with RBC lysis solution (Quiagen, Cat: 158904). 9 × 105 splenocytes or lymph node cells were seeded in triplicate into 96-well MSIPN4W multiscreen plates (Millipore, Cat: MSIPN4W50) coated with 6 μg/mL anti-mouse interferon gamma (IFNγ) antibody (AN18 monoclonal capture antibody, Mabtech, Cat: 3321-2-1000). The cells were stimulated with 5 μM of peptide (treatment), DMSO (negative control), or concanavalin A (positive control, Sigma-Aldrich, Cat: C5275). Incubation with the presence of the antigen lasted from 16 to 18 h after which, they were washed off and secondary antibody was added (monoclonal anti-mouse IFNγ R4-8A2 biotinylated antibody, Mabtech, Cat: 3321-6-10), followed by streptavidin-ALP (Mabtech, Cat: 3321–10) for detection. The assay was developed with BCIP/NBT plus substrate (Mabtech, Cat: 3650–10). Spots of IFNγ secreted from activated cells were counted with the ELISpot reader, CTL ImmunoSpot S6 Ultimate analyzer (Cellular Technology Limited), and Immunospot Software (Cellular Technology Limited).
Lecoq I., Kopp K.L., Chapellier M., Mantas P., Martinenaite E., Perez-Penco M., Rønn Olsen L., Zocca M.B., Wakatsuki Pedersen A, & Andersen M.H. (2022). CCL22-based peptide vaccines induce anti-cancer immunity by modulating tumor microenvironment. Oncoimmunology, 11(1), 2115655.
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10

ELISPOT Assay for Influenza Peptide Immunogenicity

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The ELISPOT assay was performed as previously described24 (link). Briefly, splenocytes were prepared from immunized mice using a 40-µm cell strainer and the red blood cells were lysed using and ACK lysis buffer. The splenocytes were washed and resuspended in cRPMI-10% FBS. The splenocytes from groups of 5 immunized mice were pooled. The splenocytes were mixed with peptide pools and added to 96 well Immunlon-P filter plates (Millipore) that had been coated with anti-mouse interferon-γ AN18 antibody (MABTECH). The plates were incubated at 37 °C with 5% CO2 overnight. The plates were washed and mouse interferon-γ was detected using R4-6A2 antibody and streptavidin-ALP (MABTECH). The spots were developed using BCIP/NBT substrate (Moss). The peptides consisted of 17-mers overlapping by 12 amino acids (Genscript). A total of 258 peptides were included in the pools and the sequences represented included, A/TX/1/77, A/Aichi/2/68, A/Mississippi/1/85 and the H3-con protein.
Lingel A., Bullard B.L, & Weaver E.A. (2017). Efficacy of an Adenoviral Vectored Multivalent Centralized Influenza Vaccine. Scientific Reports, 7, 14912.
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