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Np25 bsa

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NP25-BSA is a laboratory product manufactured by Biosearch Technologies. It is a nanoparticle-based reagent containing bovine serum albumin (BSA). The core function of NP25-BSA is to serve as a standard or reference material for various analytical and research applications.

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Lab products found in correlation

Np4 bsa, by Biosearch Technologies (4 mentions) 96 well immune plate, by Thermo Fisher Scientific (3 mentions) P nitrophenylphosphate, by Southern Biotech (3 mentions) Np5 bsa, by Biosearch Technologies (3 mentions) Mouse igm and igg3, by Southern Biotech (2 mentions) Ap conjugated anti mouse igm and igg3 antibodies, by Southern Biotech (2 mentions) Tmb peroxidase substrate kit, by Bio-Rad (2 mentions) H3n2 ha or h3n2 na, by Sino Biological (2 mentions) Hrp conjugated secondary antibodies against mouse igg and iga, by Southern Biotech (2 mentions) Anti mouse ig h l, by Southern Biotech (1 mentions) Np9 bsa, by Biosearch Technologies (1 mentions) Nbt bcip, by Roche (1 mentions) Quanta lite ana elisa, by Inova Diagnostics (1 mentions) High binding 96 well plate, by Corning (1 mentions) 1 step abts substrate solution, by Thermo Fisher Scientific (1 mentions) Tween 20, by Merck Group (1 mentions) Immulon 4hbx, by Thermo Fisher Scientific (1 mentions) Anti mouse igg1 hrp, by Southern Biotech (1 mentions) Fluostar optima plate reader and software, by BMG Labtech (1 mentions) Streptavidin hrp, by BD (1 mentions)

13 protocols using np25 bsa

1

ELISA and ELISPOT Assays for Antibody Responses

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For SRBC immunization experiments, 96-well immune plates (Thermo Fisher Scientific) were coated with anti–mouse Ig(H+L) (Southern Biotech). For NP immunization experiments, plates were coated with NP9-BSA or NP25-BSA (Biosearch Technologies). Mouse serum samples were incubated for 2 h at room temperature. Standard curves were generated using mouse IgM and mouse IgG1 (Southern Biotech). Bound antibodies were detected by AP-conjugated anti–mouse IgM and anti–mouse IgG1 antibodies (Table S5). Plates were developed with p-nitrophenylphosphate (Southern Biotech) dissolved in substrate buffer. For ELISPOT analysis, the spleens and BM of NP-KLH–immunized mice were removed at the indicated time points, and single cell suspensions were subjected to hypotonic lysis. The samples were plated overnight on 96-well filtration plates (Millipore) coated with NP25-BSA (Biosearch Technologies). The cells were plated in 1:2 serial dilutions, starting at 8 × 105 cells for BM-derived samples, and at 105 cells for spleen-derived fractions. NP-specific IgG1-secreting cells were detected by AP-conjugated anti–mouse IgG1 antibody (Table S5). Plates were developed with nitro blue tetrazolium chloride-5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche).
Heise N., De Silva N.S., Silva K., Carette A., Simonetti G., Pasparakis M, & Klein U. (2014). Germinal center B cell maintenance and differentiation are controlled by distinct NF-κB transcription factor subunits. The Journal of Experimental Medicine, 211(10), 2103-2118.
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2

Measurement of Antibody Binding to NP-BSA Conjugates

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Autoreactivity against nuclear and cytoplasmic self-antigens was determined with QUANTA Lite ANA ELISA (Inova Diagnostics) as described (52 (link)). Polyreactivity against ssDNA, dsDNA, Keyhole limpet hemocyanin (KLH), human insulin and lipopolysaccharide (LPS) was determined as described (22 (link)). To measure NP binding, high-binding 96-well plates (Corning) were coated overnight with 50 μl of PBS containing 10 μg/ml of NP4-BSA or NP25-BSA (Biosearch Technologies). After washing with PBS containing 0.05% of Tween 20 (Sigma), wells were blocked with PBS containing 1% of BSA for 2 h at room temperature. Monoclonal antibodies were incubated at 4 μg/ml or 7 consecutive 1:4 dilutions in PBS for 2 h at room temperature. After washing, HRP-conjugated goat α-human IgG (Jackson ImmunoResearch) was added at 0.16 μg/ml for 1 h at room temperature. After additional washing, HRP was revealed with 1-Step ABTS Substrate Solution (ThermoFisher Scientific). Absorbance was measured at 405 nm after incubation for 20 min at room temperature.
Mayer C.T., Gazumyan A., Kara E.E., Gitlin A.D., Golijanin J., Viant C., Pai J., Oliveira T.Y., Wang Q., Escolano A., Medina-Ramirez M., Sanders R.W, & Nussenzweig M.C. (2017). The Microanatomic Segregation of Selection by Apoptosis in the Germinal Center. Science (New York, N.Y.), 358(6360), eaao2602.
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3

NP-specific IgG1 Response in Mice

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WT or Esrrg-cKO mice, 2–3 months old, were immunized with 100 μg NP23-KLH in alum (1:1) at week 0 and boosted at week 2. Serum samples were collected at weeks –1, 1, and 3. Serum NP-specific IgG1 were determined as previously described (13 (link)) by ELISA using plates coated with NP4-BSA or NP25-BSA (Biosearch Technologies), followed by incubation with 1:500 diluted serum samples and developed with alkaline phosphatase–conjugated goat anti-mouse IgG1. All samples were run in duplicate.
Li W., Gong M., Park Y.P., Elshikha A.S., Choi S.C., Brown J., Kanda N., Yeh W.I., Peters L., Titov A.A., Teng X., Brusko T.M, & Morel L. (2021). Lupus susceptibility gene Esrrg modulates regulatory T cells through mitochondrial metabolism. JCI Insight, 6(14), e143540.
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4

NP-BSA Immunization Antibody Assay

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For NP-LPS and NP-Ficoll immunization experiments, 96 well immune-plates (Thermo Fisher Scientific) were coated with NP25-BSA (Biosearch Technologies). Mouse serum samples were incubated for 2h at RT. Standard curves were generated using mouse IgM and IgG3 (Southern Biotech). Bound antibodies were detected by AP-conjugated anti-mouse IgM and IgG3-antibodies (Southern Biotech). Plates were developed with p-nitrophenylphosphate (Southern Biotech) dissolved in substrate buffer.
Milanovic M., Heise N., De Silva N.S., Anderson M.M., Silva K., Carette A., Orelli F., Bhagat G, & Klein U. (2016). Differential requirements for the canonical NF-κB transcription factors c-REL and RELA during the generation and activation of mature B-cells. Immunology and cell biology, 95(3), 261-271.
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5

Measurement of NP-specific IgG1 by ELISA

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NP-specific IgG1 was measured by ELISA. In brief, NP25-BSA (Biosearch Technologies) was coated on ELISA plates (Immulon 4HBX; Thermo Fisher) overnight. The plates were then washed with ELISA wash buffer (0.5% Tween in PBS), serially diluted sera were applied to the plates, and plates were incubated 2 h at room temperature. Anti–mouse IgG1 HRP (Southern Biotech) was used to detect NP-specific IgG1. To measure IgG1 affinity maturation, antibody titer was determined with NP4-BSA and NP25-BSA. For allotype-specific IgG1 detection, anti–mouse IgG1a or IgG1b-biotin antibody (BD Biosciences) was used and followed by a streptavidin-HRP (BD Biosciences) reaction. After a wash step, 2,2'-Azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) substrate (KPL) was added to the wells, and enzyme reaction was stopped by ABTS HRP Stop Solution (KPL). Optical density at 405 nm was measured with Fluostar Optima plate reader and software (BMG Labtech).
Watanabe M., Fujihara C., Radtke A.J., Chiang Y.J., Bhatia S., Germain R.N, & Hodes R.J. (2017). Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells. The Journal of Experimental Medicine, 214(9), 2795-2810.
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6

NP and LCMV Antibody ELISA Assays

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For NP specific ELISPOTS, splenocytes were incubated on 10 ug/ml of NP5-BSA (high affinity) or NP25-BSA (all affinities) (Biosearch Technologies) coated plates (Millipore) and incubated with biotin-anti-mouse-IgG1or IgM (Southern Biotech) followed by incubation with ExtrAvidin-Alkaline Phosphatase (Sigma) and developed with NBT/BCIP substrate (Sigma). Spots were enumerated on CTL-ImmunoSpot reader (Cellular Technologies). LCMV specific antibodies were detected in serum by ELISA. Lysate from baby hamster kidney (BHK) cells infected with LCMV Armstrong was used to coat ELISA plates. HRP-linked antibodies against mouse IgG were used to detect the LCMV reactive antibodies. Relative OD values were determined at 450 nm, and values at dilutions within a linear range were used to determine final relative absorption.
Barnett L.G., Simkins H.M., Barnett B.E., Korn L.L., Johnson A.L., Wherry E.J., Wu G.F, & Laufer T.M. (2014). B cell antigen presentation in the initiation of Follicular Helper T cell and Germinal Center differentiation. Journal of immunology (Baltimore, Md. : 1950), 192(8), 3607-3617.
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7

Measurement of Anti-NP and Influenza Antibodies

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Sera with serial dilutions were added to 96-well plates coated with 5 μg ml−1 NP5-BSA (for detecting high-affinity anti-NP) or NP25-BSA (for detecting both high- and low-affinity anti-NP) obtained from Biosearch Technologies and incubated at room temperature for 2 h, followed by incubation with HRP-conjugated secondary antibodies against mouse IgM, IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotechnology). The plates were developed with TMB peroxidase substrate kit (Bio-Rad Laboratories, Hercules, CA) and optical densities at 450 nm were measured. A mixture of sera from wild type mice immunized with NP-KLH was used to establish standard curves in each plate and antibody levels were shown as relative titers. Influenza A H3N2 specific antibodies in the sera or BAL fluid were measured similarly as above except that 96-well plates were coated with 2 μg ml−1 H3N2 HA or H3N2 NA (Sino Biological) and HRP-conjugated secondary antibodies against mouse IgG and IgA (Southern Biotechnology) were used.
Chen M., Hong M.J., Sun H., Wang L., Shi X., Gilbert B.E., Corry D.B., Kheradmand F, & Wang J. (2014). Essential Role for Autophagy in the Maintenance of Immunological Memory Against Influenza Infection. Nature medicine, 20(5), 503-510.
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8

Quantification of Antigen-Specific Antibodies

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To determine antigen-specific serum IgM, IgG1 and IgG3 titers, 96 well flat-bottom Nunc MaxiSorp Microwell plates were coated overnight with 50 μg /ml NP25-BSA (Biosearch Technologies, Novato, CA) diluted in carbonate buffer. The plates were blocked with 1% BSA for 2 h at room temperature prior to the addition of serially diluted serum samples. The serum samples were diluted with 1% BSA and two-fold dilutions were performed: 1/800 to 1/6400 for IgM, 1/10000 to 1/80000 for IgG1, and 1/200 to 1/3200 for IgG3. Subsequently, HRP-conjugated anti-IgM, anti-IgG1 or anti-IgG3 were added to the wells and NP-specific Ig levels were detected using 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS) substrate (Southern Biotechnology, Birmingham, AL). Absorbance was measured at 405 nm.
Ray A., Yuan C.Y., Miller N.M., Mei H, & Dittel B.N. (2015). 2B4 Is Dispensable for T-Dependent B Cell Immune Responses, but Its Deficiency Leads to Enhanced T-Independent Responses Due to an Increase in Peritoneal Cavity B1b Cells. PLoS ONE, 10(8), e0137314.
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9

Assessing Antibody Affinity to NP Haptens

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Serum was collected from mice on day 7 or 9. High binding plates (Costar) were coated overnight at 4°C with [NP(4)-BSA] or [NP(25)-BSA] (50 μg/ml, Biosearch Technologies). Horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (Amersham) was used for detection. Relative affinity of the NP-specific IgG antibodies was calculated from the ratio of antibody binding to low-density hapten [NP(4)-BSA] versus high-density hapten [NP(25)-BSA] coated plates.
Wang N., Halibozek P.J., Yigit B., Zhao H., O’Keeffe M.S., Sage P., Sharpe A, & Terhorst C. (2015). Negative Regulation of Humoral Immunity Due to Interplay between the SLAMF1, SLAMF5, and SLAMF6 Receptors. Frontiers in Immunology, 6, 158.
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10

Quantifying NP-Specific IgG1 ASCs in Mice

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The numbers of NP-specific IgG1 ASCs in the BM of NP-OVA–immunized WT and Bhlhe40−/− mice 26 d after immunization were determined by ELISPOT assay. To this end, MAIPSWU10 96-well plates (Millipore) were coated overnight at 4°C with 5 µg/ml NP25-BSA (BioSearch Technologies) in PBS. RBC-depleted BM cells were plated in duplicate threefold dilution series from 106 to 1.23 × 105 cells per well and incubated overnight at 37°C in RPMI supplemented with 10% FCS, 1% L-glutamine, 1% penicillin-streptomycin, and 0.1% β-mercaptoethanol. Plates were washed with PBS/0.05% Tween-20 and subsequently incubated with 1 µg/ml biotinylated anti-mouse IgG1 antibody (RMG1-1; BioLegend). Spots were visualized with alkaline phosphatase–conjugated streptavidin (Mabtech) and BCIP-NBT-plus substrate (Mabtech). Plates were extensively washed, spots were counted using an AID ELISpot Reader (AID Diagnostika), and the mean cell number of NP-specific IgG1 ASCs per 106 BM cells was calculated from each threefold dilution series.
Rauschmeier R., Reinhardt A., Gustafsson C., Glaros V., Artemov A.V., Dunst J., Taneja R., Adameyko I., Månsson R., Busslinger M, & Kreslavsky T. (2021). Bhlhe40 function in activated B and TFH cells restrains the GC reaction and prevents lymphomagenesis. The Journal of Experimental Medicine, 219(2), e20211406.
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