Tnp bsa

Manufactured by Biosearch Technologies

Sourced in United States, Austria

TNP-BSA is a protein conjugate composed of 2,4,6-trinitrophenyl (TNP) covalently linked to bovine serum albumin (BSA). It is commonly used as a hapten carrier in immunological studies.

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Lab products found in correlation

Tnp ficoll, by Biosearch Technologies (3 mentions) Ovalbumin (ova), by Merck Group (3 mentions) Streptavidin hrp, by Thermo Fisher Scientific (2 mentions) Dnp hsa, by Merck Group (2 mentions) Np bsa, by Biosearch Technologies (2 mentions) Tnp65 ficoll, by Biosearch Technologies (1 mentions) Nunc maxisorp, by Thermo Fisher Scientific (1 mentions) Opteia tmb substrate reagent set, by BD (1 mentions) Sunrise photometer, by Tecan (1 mentions) Immobilon p membrane, by Merck Group (1 mentions) 3 amino 9 ethylcarbazole, by Merck Group (1 mentions) Aid ispot reader system, by Autoimmun Diagnostika (1 mentions) Streptavidin horseradish peroxidase, by Agilent Technologies (1 mentions) Igg3 r40 82, by BD (1 mentions) Countess 2 fl, by Thermo Fisher Scientific (1 mentions) Interleukin 3 (il 3), by American Type Culture Collection (1 mentions) C57bl 6 mice, by Charles River Laboratories (1 mentions) N n dimethylformamide (dmf), by Merck Group (1 mentions) Elisa plate, by Thermo Fisher Scientific (1 mentions) Super aqua blue substrate, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

TNP-conjugated BSA (5 citations)

14 protocols using tnp bsa

TNP-Ficoll B Cell Expansion in Mice

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TNP-Ficoll-specific B cell expansion experiments were performed on mice immunized intraperitoneally (i.p.) with 50 μg TNP₆₅-Ficoll (Biosearch Technologies, Novato, CA) as previously described (18 (link)). For serum Ab analyses, mice were immunized i.p. with 25 μg of TNP₆₅-Ficoll. Ags were diluted in sterile PBS and injected in a final volume of 200 μl. In some experiments, mice were administered 250 μg MB22-10 mAb (39 (link)) or control mouse IgG2a (Southern Biotechnology Associates, Birmingham, AL) in 200 μl sterile PBS i.p. the day before immunization.
ELISAs were as previously described (18 (link), 19 (link), 40 (link), 41 ). Sera were diluted 1:1000 for IgM detection and 1:500 for IgG detection in TBS containing 1% BSA. TNP-specific Ab levels were measured by adding diluted serum samples to Costar plates that had been coated with 5 μg/ml TNP-BSA (Biosearch Technologies) in 0.1 M borate buffered saline overnight at 4°C. PPS-3-specific ELISAs used Nunc Maxisorp plates that were coated at room temperature with 5 μg/ml PPS-3 (ATCC) overnight, blocked with TBS containing 1% BSA, and incubated with serum that had been pre-blocked with 10 μg/ml cell wall polysaccharide (Serum Statens Institute). AP-conjugated polyclonal goat anti-mouse IgM, IgG3, and IgG Abs (all from Southern Biotechnology Associates) and pNPP (Sigma) were used to detect Ag-specific Ab.

Haas K.M., Johnson K.L., Phipps J.P, & Do C. (2018). CD22 promotes B-1b cell responses to T cell-independent type 2 antigens. Journal of immunology (Baltimore, Md. : 1950), 200(5), 1671-1681.

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Quantification of OVA-specific IgG by ELISA

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OVA-specific serum IgG was measured by ELISA and normalized to a standard serum (arbitrary units, AU). 96-well microtiter plates (Nunc MaxiSorp^™, Affymetrix eBioscience, Santa Clara, CA, USA) were coated with 0.5 μg OVA per well (Sigma-Aldrich, St. Louis, MO, USA) in coating buffer (Candor Bioscience GmbH, Wangen, Germany) overnight at 4°C, washed with PBS/0.05% Tween20^™ and blocked with PBS/0.05% Tween20^™/10% FCS. IgG binding was detected using goat anti-mouse IgG coupled to HRP (Southern Biotech, Birmingham, AL, USA) and BD OptEIATM TMB Substrate Reagent Set (BD, Franklin Lakes, NJ, USA). Optical density at 450 nm was measured with the Tecan Sunrise photometer (Tecan Group Ltd., Maennedorf, Switzerland).
The relative affinity of TNP-specific IgG antibodies was determined based on the quantification of binding to differently haptenized bovine serum albumin (BSA)–TNP₂-BSA and TNP₂₆-BSA (adapted from [34 (link)])–as the carrier protein. IgG binding to TNP-BSA (Biosearch Technologies, Petaluma, CA, USA) was measured by ELISA as described above, and relative affinity was calculated as the ratio of the binding to TNP₂ to TNP₂₆.

Schmoeckel K., Mrochen D.M., Hühn J., Pötschke C, & Bröker B.M. (2018). Polymicrobial sepsis and non-specific immunization induce adaptive immunosuppression to a similar degree. PLoS ONE, 13(2), e0192197.

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ELISPOT for Trout B Cell Enumeration

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ELISPOT was used to quantify the number of total and TNP-specific IgM-secreting B cells in spleen and kidney of experimental fish. For this, ELISPOT plates containing Immobilon-P membranes (Millipore) were activated with 70% ethanol and coated with 2 μg/ml of an anti-trout IgM mAb (clone 4C10) or with 5 μg/ml of TNP-BSA (Biosearch Technologies) overnight at 4 °C in agitation. Non-specific binding sites were blocked by incubation with 2% BSA in PBS for 2 h at RT. After that, leukocytes from rainbow trout kidney and spleen were added to the wells in triplicate at a concentration of 5 × 10⁴ cells per well. After 24 h of incubation at 20 °C, cells were washed 5 times with PBS and plates blocked with 2% BSA in PBS for 1 h at RT. After blocking, biotinylated anti-trout IgM mAb (clone 4C10) was added to the plates (1 μg/ml) that were subsequently incubated for 1 h at RT. Following additional washing steps (5 times in PBS), the plates were developed using streptavidin-HRP (Thermo Fisher Scientific) (100 ng/ml) for 1 h at RT, washed again with PBS and incubated with 3-amino 9-ethylcarbazole (Sigma-Aldrich) for 30 min at RT in the dark. The substrate reaction was stopped by washing the plates with tap water. Once the membranes were dried, the number of spots in each well was determined using an AID iSpot Reader System (Autoimmun Diagnostika GMBH).

Martín-Martín A., Simón R., Abós B., Díaz-Rosales P, & Tafalla C. (2020). Rainbow trout mount a robust specific immune response upon anal administration of thymus-independent antigens. Developmental and Comparative Immunology, 109, 103715.

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Determining Serum Immunoglobulin Concentrations

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To determine the concentration of basal serum immunoglobulins, ninety-six-well plates (BD Biosciences) were coated with antibody (IgM (II/41), IgG1 (A85-3), IgG2a (R11-89, IgG2b (R9-91), and IgG3 (R2-38), all from BD Biosciences) in PBS/Tris-HCl 0.1M at 5 μg/ml overnight at 4ºC. Isotype standards Abs were obtained from Sigma-Aldrich. TNP-BSA (Biosearch Technologies) was used to coat plates for detecting TNP-specific Ig in the sera of immunized mice. After blocking with PBS and 1% BSA, dilutions of mouse serum were added, and again plates were incubated overnight at 4ºC. The following day, biotinylated anti-mouse IgM (R60.2), IgG1 (A85.1), IgG2a (R19-15), IgG2b (R12-3), and IgG3 (R40-82) secondary antibodies (BD Biosciences) were added and plates were incubated for 1 h at room temperature. After washing, the plates were incubated with streptavidin-horseradish peroxidase (Dako) for 1 h at room temperature. Following washing, 3,3′,5,5′-Tetramethylbenzidine (Sigma-Aldrich) was added and the plates were read at 655 nm.

Galindo-Campos M.A., Bedora-Faure M., Farrés J., Lescale C., Moreno-Lama L., Martínez C., Martín-Caballero J., Ampurdanés C., Aparicio P., Dantzer F., Cerutti A., Deriano L, & Yélamos J. (2019). Coordinated signals from the DNA repair enzymes PARP-1 and PARP-2 promotes B-cell development and function. Cell Death and Differentiation, 26(12), 2667-2681.

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Bone Marrow-Derived Mast Cell Activation

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Primary mast cell cultures were established by isolating bone marrow from the tibia and femur of wild-type C57BL/6 mice (Charles River) and inducing differentiation with IL-3 (from Wehi-3b cells, American Type Culture Collection – ATCC) and PGE₂ (Sigma) conditioned culture media. The BMMCs were cultured twice weekly by collection, centrifugation, and resuspension of cells in fresh media, with cell viability confirmed by NucBlue stain (Life Technologies, R37605) and cell growth measured on a Countess II FL (Life Technologies Inc., AMQAF1000). Cells were maintained at a density of 0.5 × 10⁶ cells per mL and incubated at 37 °C with 5% carbon dioxide (CO₂). For IgE-mediated activation, BMMCs were sensitized overnight with TNP-specific IgE harvested from TIB-141 cells (ATCC) and the following day unbound IgE was washed away with RPMI 1640. Prior to stimulation, the cells were resuspended in RPMI 1640 supplemented with 10% FBS and 1% pen/strep. Cells were then separated for various time points (15 min, 30 min, 1 h, 3 h, 6 h, 24 h) and stimulated with 100 ng mL⁻¹ TNP-BSA (Biosearch Technologies, Novato, CA) under SCF potentiation at 100 ng mL⁻¹ (Peprotech, Dollard des Ormeaux, QC).

Li Y., Mansour H., Watson C.J., Tang Y., MacNeil A.J, & Li F. (2021). Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay. Chemical Science, 12(6), 2133-2137.

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Intradermal IgE-mediated Anaphylaxis Assay

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Age- and sex-matched mice were randomized allocating to experimental group. 20 ng monoclonal mIgE specific for OVA, DNP (clone SPE-7; Sigma-Aldrich), or TNP (clone IgE-3; BD) or 5 ng polyclonal IgE specific for OVA or peanut extracts was injected intradermally in the BALB/c mice ears, and the next day mice were intravenously challenged with PBS containing 125 µg OVA (Sigma-Aldrich), peanut extract, DNP-HSA (Sigma-Aldrich), or TNP-BSA (conjugation ratio 13; Biosearch Technologies) and 2% Evans blue dye in PBS. 45 min after challenge, the mice were sacrificed and the ears were excised and minced before incubation in N,N-dimethylformamide (EMD Millipore) at 55°C for 3 h. The degree of blue dye in the ears was quantitated by the absorbance at 595 or 650 nm. For experiments of hIgE, 250 ng αOVA-hIgE was injected intradermally in hFcεRIα⁺/mFcεRIα^−/− or mFcεRIα^−/− mice, followed by intravenous administration of OVA and Evans blue dye 4 h later.

Shade K.T., Platzer B., Washburn N., Mani V., Bartsch Y.C., Conroy M., Pagan J.D., Bosques C., Mempel T.R., Fiebiger E, & Anthony R.M. (2015). A single glycan on IgE is indispensable for initiation of anaphylaxis. The Journal of Experimental Medicine, 212(4), 457-467.

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Quantifying T Cell-Dependent and Independent Antibody Responses

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Itpkb^+/+ and Itpkb^fl/fl mice were immunized intraperitoneal (i.p.) with either 100 μl DNP-KLH/Alum (Calbiochem) at 1mg/ml to assess T cell-dependent antibody responses, or with 100 μl TNP-(27)-AECE-Ficoll (BioSearch Technologies) to assess T cell-independent responses. Blood was obtained from mice before injection and post-immunization on day 9 for DNP-KLH and day 11 for TNP-Ficoll. Serum was analyzed by enzyme-linked immunosorbent assay (ELISA). ELISA plates (NUNC) were coated overnight at 4°C with TNP-BSA (BioSearch Technologies) at 50 μg/ml bi-carbonate buffer. Plates were washed 3 times (0.05% Tween/PBS), blocked with 1% BSA/PBS for 2 h at 25°C, then serial dilutions of serum samples were added, followed by incubation for 2 h at 25°C. Plates were washed six times before adding anti-IgG1-HRP (US Biological, 1/2,000) or anti-IgG3-HRP (Jackson Lab, 1/5000) for 1 h at room temperature. Plates were washed six times, antibodies were detected with Super Aqua Blue substrate (eBioscience), stopped with 0.625 M Oxalic Acid (Fluka) and absorbance was measured at 405 nm.

Miller A.T., Dahlberg C., Sandberg M.L., Wen B.G., Beisner D.R., Hoerter J.A., Parker A., Schmedt C., Stinson M., Avis J., Cienfuegos C., McPate M., Tranter P., Gosling M., Groot-Kormelink P.J., Dawson J., Pan S., Tian S.S., Seidel H.M, & Cooke M.P. (2015). Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease. PLoS ONE, 10(6), e0131071.

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Trout IgM ELISA Protocol

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The presence of TNP-specific IgM in serum was estimated by ELISA as previously described (27 (link)). For this, microtitre plates were coated with 5 μg/ml of TNP-BSA (Biosearch technologies) in a volume of 100 μl PBS overnight at 4°C. Thereafter, non-specific binding sites were blocked by incubation with 1% BSA in PBS with 0.05% Tween 20 (PBT) for 1 h at RT. Plates were then washed with PBT and a 1/500 dilution of each serum sample in PBS 1% BSA added to each well and incubated for 1 h at RT. Serum samples from all groups were analyzed in duplicate wells. After washing three times with PBT, each well was incubated with 1 μg/ml biotinyilated anti-trout IgM mAb (clone 4C10) diluted in PBS 1% BSA for 1 h at RT. The plates were washed again three times in PBT and 100 ng/ml of HRP-streptavidin (Thermo Fisher Scientific) added to each well in 100 μl PBS 1% BSA. After incubation at RT for 1 h, 100 μl of o-phenylenediamine dihydrochloride substrate reagent (Sigma-Aldrich) were added to each well. The reaction was stopped after 15 min by adding 50 μl of 2.5 M H₂SO₄. Absorbances were recorded at 490 nm using a FLUOstar Omega (BMG Labtech) plate reader. Internal positive and negative control samples were also included.

Martín D., Ordás M.C., Carvalho I., Díaz-Rosales P., Nuñez-Ortiz N., Vicente-Gil S., Arrogante A., Zarza C., Machado M., Costas B, & Tafalla C. (2023). L-methionine supplementation modulates IgM+ B cell responses in rainbow trout. Frontiers in Immunology, 14, 1264228.

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Thymus-Independent and -Dependent Immune Responses

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Control (Ptpn1^f/f and mb1cre) and Ptpn1^f/f-mb1cre mice were immunized with 20 µg thymus-independent antigen TNP-Ficoll (Biosearch Technologies) intraperitoneally. Mice were bled before and 7 d after immunization. To determine the thymus-dependent response, control (Ptpn1^f/f or mb1cre) and Ptpn1^f/f-mb1cre were immunized with 20 µg TNP-BSA (Biosearch Technologies) precipitated with alum as adjuvant and injected intraperitoneally. At day 21, a second immunization (boost) was performed with 20 µg TNP-BSA. Mice were bled before immunization and on day 14, 21 (before the boost), 28, and 35 after injection.

Medgyesi D., Hobeika E., Biesen R., Kollert F., Taddeo A., Voll R.E., Hiepe F, & Reth M. (2014). The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity. The Journal of Experimental Medicine, 211(3), 427-440.

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Quantification and Verification of IgE Antibodies

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Mouse or human IgE was quantified by sandwich ELISA according to instructions from mouse or human IgE ELISA quantitation sets (Bethyl Laboratories, Inc.). Antibodies specific for OVA were verified by 96-well Nunc plates plate coated with 75 µg/ml OVA (Sigma-Aldrich), blocked with 2% BSA in PBS, and probed with goat polyclonal anti–mouse or anti–human IgE-HRP (2 ng/ml; Bethyl Laboratories, Inc.). A similar protocol was used for verifying antibodies specific for DNP and TNP, except 5 µg/ml DNP-HSA (Sigma-Aldrich) and TNP-BSA (conjugation ratio 13; Biosearch Technologies) was used for coating. All reactions were detected by 3,3,5,5-tetramethylbenzidine (TMB; Thermo Fisher Scientific) and stopped by 2 M sulfuric acid, and the absorbance was measured at 450 nm.

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