Streptavidin alkaline phosphatase

Manufactured by Roche

Sourced in United States, Germany

Streptavidin–alkaline phosphatase is a conjugate of the protein streptavidin and the enzyme alkaline phosphatase. It is used in various bioanalytical and diagnostic applications that involve the detection and quantification of target analytes.

Automatically generated - may contain errors

Lab products found in correlation

4 nitrophenyl phosphate, by Merck Group (4 mentions) Np30 bsa, by Biosearch Technologies (2 mentions) Versamax, by Molecular Devices (2 mentions) Alkaline phosphatase substrate solution, by Merck Group (2 mentions) Np36 cgg, by Biosearch Technologies (1 mentions) Versamax microplate reader, by Molecular Devices (1 mentions) Biotin conjugated anti igg, by Southern Biotech (1 mentions) 7 b6 1 biotin, by Mabtech (1 mentions) Nbt 5 bromo 4 chloro 3 indolyl phosphate, by Merck Group (1 mentions) Streptavidin alkaline phosphatase, by Merck Group (1 mentions) Mahas4510, by Merck Group (1 mentions) Ctl immunospot analyzer, by Cellular Technology (1 mentions) Mouse ifn gamma quantikine elisa kit, by R&D Systems (1 mentions) Biotinylated anti ifn γ antibody, by Thermo Fisher Scientific (1 mentions) Bcip nbt substrate, by Mabtech (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions) Multiscreen ip plate, by Merck Group (1 mentions) 5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium substrate, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Roche (1 mentions) Np5 bsa, by Biosearch Technologies (1 mentions)

9 protocols using streptavidin alkaline phosphatase

NP-CGG Antibody Response Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antigens for immunization were prepared by mixing NP₃₆-CGG (Biosearch Technologies) dissolved in PBS and 10% KAl(SO4)₂ at 1:1 ratio and adjusting pH to 7, to form precipitate. Five micrograms of NP-CGG precipitated in alum was injected intraperitoneally for NP-specific antibody responses.
Microtitre plates were coated with the following: (i) 10 μg ml⁻¹ NP₃₀-BSA (Biosearch Technologies) in PBS, for measurement of NP-specific antibody, or (ii) 2.5 μg ml⁻¹ goat anti-mouse κ- and goat anti-mouse λ-antibodies diluted in PBS containing Ca²⁺ and Mg²⁺, pH 7.0–7.2, for measurement of total serum antibodies. Nonspecific binding was blocked with 0.5% BSA in PBS. Serum samples were serially diluted in 0.5% BSA in PBS and were incubated in blocked plates overnight at 4 °C. Plates were incubated for 2 h with biotin-conjugated anti-IgM (1020-08, 1 μg ml⁻¹, Southern Biotech), anti-IgG1 (1070-08, 1 μg ml⁻¹, Southern Biotech) or anti-IgG (1030-08, 1 μg ml⁻¹, Southern Biotech), for 1 h with streptavidin–alkaline phosphatase (Roche), and then with alkaline phosphatase substrate solution containing 4-nitro-phenyl phosphate (Sigma) for colour development, followed by quantification on a VERSAmax microplate reader (Molecular Devices).

Lai M., Gonzalez-Martin A., Cooper A.B., Oda H., Jin H.Y., Shepherd J., He L., Zhu J., Nemazee D, & Xiao C. (2016). Regulation of B-cell development and tolerance by different members of the miR-17∼92 family microRNAs. Nature Communications, 7, 12207.

+ Open protocol

+ Expand

LCMV Antibody Detection Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Microtiter plates were coated with lysate of LCMV Cl13–infected baby-hamster kidney (BHK) cells. Nonspecific binding was blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Serum samples were serially diluted in PBS with 1% BSA and incubated in blocked plates at 4°C overnight. Plates were incubated with biotin-conjugated anti-IgG (1030-08; Southern Biotech) for 2 hours, with streptavidin–alkaline phosphatase (Roche) for 1 hour, and then with alkaline phosphatase substrate solution containing 4-nitro-phenyl phosphate (Sigma-Aldrich) for color development, followed by quantification on a VERSAmax microplate reader (Molecular Devices).

Huang Z., Kang S.G., Li Y., Zak J., Shaabani N., Deng K., Shepherd J., Bhargava R., Teijaro J.R, & Xiao C. (2021). IFNAR1 signaling in NK cells promotes persistent virus infection. Science Advances, 7(13), eabb8087.

+ Open protocol

+ Expand

IFN-γ ELISpot Assay Protocol

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Enzyme-linked immunospot (ELISpot) flat-bottomed, 96-well nitrocellulose plates (MAHA S4510, Millipore) were coated with IFN-γ mAb (2 μg ml⁻¹, 1-D1K; MABTECH) and incubated for 2 h at 37 °C. After washing with PBS, plates were blocked with 10% human AB serum for 2 h at 37 °C. Cells were washed, concentrated, plated into each well titrating down in twofold serial dilutions starting from 400,000 PBMCs, in the presence of peptides, negative control (DMSO) and positive control (phorbol 12-myristate 13-acetate and ionomycin (PMA)/ionomycin). Plates were incubated for 24 h in a CO₂ incubator. After incubation, the plates were semi-automatically washed with PBS and then IFN-γ mAb (0.2 μg ml⁻¹, 7-B6–1-biotin; MABTECH) was added to each well. After incubation for 2 h at 37 °C, plates were washed and incubated with streptavidin-alkaline phosphatase (1 μg ml⁻¹, Roche) for 1 h at room temperature. After washing unbound streptavidin-alkaline phosphatase, substrate (5-bromo-4-chloro-3-indolyl phosphate/NBT; Sigma-Aldrich) was added and incubated for 12–15 min for spots to develop. After incubation, plates were washed to remove substrate and dark-violet spots were evaluated using the CTL Immunospot analyzer and software (Cellular Technology Limited) as routinely performed in the laboratory^{38 (link)}.

Schwarz M., Torre D., Lozano-Ojalvo D., Tan A.T., Tabaglio T., Mzoughi S., Sanchez-Tarjuelo R., Bert N.L., Er Lim J.M., Hatem S., Tuballes K., Camara C., Lopez-Granados E., Paz-Artal E., Correa-Rocha R., Ortiz A., Lopez-Hoyos M., Portoles J., Cervera I., Gonzalez-Perez M., Bodega-Mayor I., Conde P., Oteo-Iglesias J., Borobia A.M., Carcas A.J., Frías J., Belda-Iniesta C., Ho J.S., Nunez K., Hekmaty S., Mohammed K., Marsiglia W.M., Carreño J.M., Dar A.C., Berin C., Nicoletti G., Noce I.D., Colombo L., Lapucci C., Santoro G., Ferrari M., Nie K., Patel M., Barcessat V., Gnjatic S., Harris J., Sebra R., Merad M., Krammer F., Kim-schulze S., Marazzi I., Bertoletti A., Ochando J, & Guccione E. (2022). Rapid, scalable assessment of SARS-CoV-2 cellular immunity by whole-blood PCR. Nature biotechnology, 40(11), 1680-1689.

+ Open protocol

+ Expand

Quantitative Ultramicro ELISA for Anti-RBD IgG

Check if the same lab product or an alternative is used in the 5 most similar protocols

Anti-RBD IgG in sera was evaluated by a quantitative ultramicro ELISA (UMELISA SARS-CoV-2 anti- RBD, Center for Immunoassay, Havana, Cuba) using d-RBD as coating antigen (4 μg/mL) and an in-house standard- characterized serum, which was arbitrarily assigned 200 AU/mL (based on a half-maximal inhibitory titer of 200 and a conventional virus neutralization titer of 160). The standard curve comprised six two-fold serial dilutions (0, 4, 8, 16, 32 and 64 AU/mL) of the standard. Samples were evaluated in duplicate. After incubation step, biotin-conjugate anti-IgG human (0.1 μg/mL) (Sigma Aldrich, San Luis, EE UU) and later, streptavidin/alkaline-phosphatase Roche, Basel, Swiss) in appropriate buffers were added. The final fluorimetric reaction was induced by adding the substrate 4-methylumbelliferyl phosphate (SLS). The reference curve was constructed using a linear interpolation function. The concentration of anti-RBD IgG was expressed as AU/mL. The seroconversion rate was calculated by dividing the concentration at each time point (at Tx) by the pre-vaccination concentration (at T0). A rate ≥ 4 was considered as seroconversion. Serum samples for this assay were extracted on days 0, 14, 42, 56, 70, 84 and 7-8 months after the last dose

Toledo-Romani M.E., García-Carmenate M., Verdecia-Sánchez L., Pérez-Rodríguez S., Rodriguez-González M., Valenzuela-Silva C., Paredes-Moreno B., Sanchez-Ramirez B., González-Mugica R., Hernández-Garcia T., Orosa-Vázquez I., Díaz-Hernández M., Pérez-Guevara M.T., Enriquez-Puertas J., Noa-Romero E., Palenzuela-Diaz A., Baro-Roman G., Mendoza-Hernández I., Muñoz Y., Gómez-Maceo Y., Santos-Vega B.L., Fernandez-Castillo S., Climent-Ruiz Y., Rodríguez-Noda L., Santana-Mederos D., García-Vega Y., Chen G.W., Doroud D., Biglari A., Boggiano-Ayo T., Valdés-Balbín Y., Rivera D.G., García-Rivera D, & Vérez-Bencomo V. (2022). Safety and immunogenicity of anti-SARS-CoV-2 heterologous scheme with SOBERANA 02 and SOBERANA Plus vaccines: Phase IIb clinical trial in adults. Med (New York, N.y.), 3(11), 760-773.e5.

+ Open protocol

+ Expand

IFN-γ ELISPOT Assay for Splenocyte Responses

Check if the same lab product or an alternative is used in the 5 most similar protocols

IFN-γ ELISPOT assay was performed by culturing 2 × 10⁵ splenocytes per well in IFN-γ ELISPOT plates (MAHAS45; Millipore, Molsheim, France). Five peptide epitopes of hSGCA with the highest probability of presentation by H-2Kb MHC class I molecules were identified by the Immune Epitope Database (http://www.iedb.org) and synthesized by GeneCust. Cells were stimulated either with one of these peptides or with 1 μM hSGCA-GST recombinant protein or with SIINFEKL peptide. As a positive control, cells were stimulated with 5 μg/mL Concanavalin A (Sigma Aldrich, St. Louis, MO, USA). After 24 h of culture at 37°C, plates were washed and the secretion of IFN-γ was revealed with a biotinylated anti-IFN-γ antibody (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), Streptavidin-Alkaline Phosphatase (Roche Diagnostics, Mannheim, Germany), and BCIP/NBT substrate (Mabtech, Les Ulis, France). Spots were counted using an AID reader (Cepheid Benelux, Leuven, Belgium) and the AID ELISpot Reader v6.0 software. Spot-forming units (SFUs) per million cells were represented after subtraction of background values obtained with unstimulated splenocytes.
IFN-γ secreted by splenocytes stimulated with the recombinant protein hSGCA-GST was measured with the Mouse IFN-gamma Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA).

Poupiot J., Costa Verdera H., Hardet R., Colella P., Collaud F., Bartolo L., Davoust J., Sanatine P., Mingozzi F., Richard I, & Ronzitti G. (2019). Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle. Molecular Therapy. Methods & Clinical Development, 15, 83-100.

+ Open protocol

+ Expand

PBMC IFN-γ ELISpot Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

96-well MultiScreen IP plates (Millipore, Billerica, MA, USA) were coated with mouse anti-swine IFN-γ mAb (clone pIFN-γ, Mabtech, Nacka Strand, Sweden) overnight at 4 °C (100 μL/well; 10 μg/mL in PBS), washed with PBS and subsequently blocked with cell culture medium (RPMI 1640 with stable glutamine supplemented with 10% [v/v] fetal calf serum, 100 IU/mL penicillin and 0.1 mg/mL streptomycin, all from PAA) for one hour at 37 °C. Per well, 3 × 10⁵ freshly isolated PBMCs were incubated with the FLUAVsw infection strain (multiplicity of infection, MOI 0.1) or mock for 24 h at 37 °C. Samples incubated in cell culture medium served as additional negative control. Thereafter, plates were washed and incubated with biotin-labeled mouse anti-IFN-γ mAbs (clone PAN, Mabtech, 100 μL/well; 0.5 μg/mL in PBS) for one hour at room temperature. This was followed by incubation with streptavidin-alkaline phosphatase (1:2000 in PBS supplemented with 0.01% Tween20 and 0.1% BSA, Roche, Mannheim, Germany) for one hour at room temperature and subsequent addition of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (100 μL/well, Sigma-Aldrich) according to manufacturer’s instructions. After intense washing and drying of plates, spots were analyzed with an AID ELISpot reader (AID, Straßberg, Germany).

Talker S.C., Koinig H.C., Stadler M., Graage R., Klingler E., Ladinig A., Mair K.H., Hammer S.E., Weissenböck H., Dürrwald R., Ritzmann M., Saalmüller A, & Gerner W. (2015). Magnitude and kinetics of multifunctional CD4+ and CD8β+ T cells in pigs infected with swine influenza A virus. Veterinary Research, 46(1), 52.

+ Open protocol

+ Expand

Serum Antibody ELISA for LCMV Detection

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Serum antibody enzyme-linked immunosorbent assays were performed as previously described [114 (link)]. Briefly, microplates were coated with LCMV-infected baby hamster kidney (BHK) cell lysates overnight. Subsequently, nonspecific binding was blocked by coating microplates with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Serial dilutions of serum were carried out in 1% BSA in PBS. After overnight incubation, plates were incubated with purified biotin-conjugated anti-mouse IgG, IgG1, or IgG2a (1030–08, 1070–08, 1080–08; Southern Biotech) antibodies for 2 hrs. Antibody detection was further performed using streptavidin–alkaline phosphatase (11089161001, Roche) for 1 hour and then alkaline phosphatase substrate solution containing 4-nitro-phenyl phosphate (N2765-50TAB, Sigma-Aldrich) for approximately 30min. A CLARIOstar Plus microplate reader was used to quantify the results.

Orozco R.C., Marquardt K., Pratumchai I., Shaikh A.F., Mowen K., Domissy A., Teijaro J.R, & Sherman L.A. (2024). Autoimmunity-associated allele of tyrosine phosphatase gene PTPN22 enhances anti-viral immunity. PLOS Pathogens, 20(3), e1012095.

+ Open protocol

+ Expand

Quantification of NP-Specific Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols

Microtiter plates were coated with NP₃₀-BSA or NP₅-BSA (10 µg/ml in PBS; LGC Biosearch Technologies) for measurement of total or high-affinity NP-specific antibody, respectively. Nonspecific binding was blocked with 0.5% BSA in PBS. Serum samples were serially diluted in 0.5% BSA in PBS and were incubated in blocked plates at room temperature for 2 h. Plates were incubated for 2 h with biotin-conjugated anti-IgM (1020-08) or anti-IgG1 (1070-08; SouthernBiotech) for 1 h with streptavidin–alkaline phosphatase (Roche) and then with alkaline phosphatase substrate solution containing 4-nitro-phenyl phosphate (Sigma-Aldrich) for color development, followed by quantification on a microplate reader (VERSAmax; Molecular Devices).

Liu W.H., Kang S.G., Huang Z., Wu C.J., Jin H.Y., Maine C.J., Liu Y., Shepherd J., Sabouri-Ghomi M., Gonzalez-Martin A., Xu S., Hoffmann A., Zheng Y., Lu L.F., Xiao N., Fu G, & Xiao C. (2016). A miR-155–Peli1–c-Rel pathway controls the generation and function of T follicular helper cells. The Journal of Experimental Medicine, 213(9), 1901-1919.

+ Open protocol

+ Expand

ELISpot and ELISA for NP-specific ASCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

NP-specific ASCs in spleen or ASCs in the iPC differentiation system were detected using an ELISpot assay on a MultiScreen 96-well filtration plate (Millipore, MSIPS4W10) coated with 10 μg/ml NP₂₉-BSA or anti-mouse kappa-UNLB and anti-mouse lambda-UNLB (2.5 µg/ml). Serially diluted cells were added to individual wells in triplicate and incubated for 6 h at 37 °C with 5% CO₂. Anti-NP IgG1, Anti-IgG1 or Anti-IgE spots were revealed by biotin-conjugated anti-mouse IgG1 or IgE Ab (Southern Biotechnology, 1070-08) in conjunction with Av-HRP and AEC substrate (Vector Laboratories, A-2004& SK-4200).
For ELISA, flat-bottom 96-well plates (NUNC, 439454) were coated with NP-BSA (10 µg/ml) or anti-mouse kappa-UNLB and anti-mouse lambda-UNLB (2.5 µg/ml) in PBS. Nonspecific binding was blocked with 0.5% BSA in PBS. Serum samples were serially diluted in 0.5% BSA in PBS and were incubated in blocked plates at room temperature for 2 h. Plates were incubated with biotin-conjugated anti-Ig (Southern Biotech) for 2 h and with streptavidin–alkaline phosphatase for 1 h (Roche, 1089161), and then incubated with alkaline phosphatase substrate solution containing 4-nitro-phenyl phosphate (Sigma-Aldrich, N2765) for color development, followed by quantification on Molecular Devices (VERSA max).

Wu J., Yang K., Cai S., Zhang X., Hu L., Lin F., Wu S.Q., Xiao C., Liu W.H, & Han J. (2022). A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation. Nature Communications, 13, 7321.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!