Avidin d hrp

Manufactured by Vector Laboratories

Sourced in United States

Avidin D-HRP is a high-affinity, tetrameric glycoprotein conjugated to horseradish peroxidase (HRP). It is used as a detection reagent in various bioassays and immunodetection techniques.

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Lab products found in correlation

Aec substrate, by Merck Group (4 mentions) Aec substrate set, by BD (3 mentions) Pvdf plate, by Merck Group (2 mentions) Goat anti mouse igg, by Jackson ImmunoResearch (2 mentions) Anti human igg biotin, by Jackson ImmunoResearch (2 mentions) Il 21, by Thermo Fisher Scientific (2 mentions) Np20 bsa, by Biosearch Technologies (1 mentions) Multi screen ha filtration, by Merck Group (1 mentions) Ba 2020, by Vector Laboratories (1 mentions) Elispot aec substrate, by BD (1 mentions) Multiscreen ha 96 well plates, by Merck Group (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Affinipure donkey anti human igg h l, by Jackson ImmunoResearch (1 mentions) Automated elispot counter, by Cellular Technology (1 mentions) Maxisorp microtiter plate, by Thermo Fisher Scientific (1 mentions) Microplate reader, by Agilent Technologies (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Tmb substrate, by BD (1 mentions) Abc elite, by Vector Laboratories (1 mentions) Biotin conjugated anti monkey igg, by Rockland Immunochemicals (1 mentions)

15 protocols using avidin d hrp

NP-Specific ELISA and ELISpot Assays

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Assays were done in Millipore Multi-Screen HA filtration 96-well plates (no. MSIPN4W). IgM, IgG1, and IgG3 coating Abs were the same as for ELISA. Secondary Abs were purchased from Vector Laboratories for detecting IgM (no. BA-2020) and IgG (no. BA-9200), followed by HRP-Avidin D (no. A-2004; Vector Laboratories). For NP-specific assays, plates were coated with NP(>20)-BSA (no. N-5050H-10; Biosearch Technologies). PBS plus 2% BSA was used as blocking buffer, ELISpot AEC substrate (no. 551951; BD Biosciences) was used to develop. Plates were scanned on a CTL ImmunoSpot S6 Entry instrument and analyzed using CTL ImmunoSpot software, version 7.0.9.5. All samples were done over a 12-step dilution series. Plotted spots per 10⁶ cells were determined by averaging spots per 10⁶ cells over a minimum of three different dilutions, with attention paid to using wells within the linear range. Counting parameters were held constant across all plates of the same assay type. Quality control of automated counting was performed on every plate by visual inspection.

Pucella J.N., Cols M., Yen W.F., Xu S, & Chaudhuri J. (2019). The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses. Journal of immunology (Baltimore, Md. : 1950), 202(5), 1383-1396.

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Enzyme-Linked Immunospot Assay for Antibodies

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Millipore Multiscreen-HA 96-well plates (Millipore #MAHA N4510) were coated with anti-mouse IgG, IgA, IgM (Rockland) diluted to 5 μg/mL in PBS and incubated overnight at 4°C. Plates were then washed with PBST (1X PBS, 0.05% Tween 20) and PBS (1X, Life Technologies) (1x PBST, 3x PBS washes) and blocked by 2 hr incubation at 37°C with cIMDM. Media was then replaced with fresh cIMDM, and counted cells from spleens, MLN, and PP were added. Plates were incubated overnight at 37°C and, following washes (4x PBS, 4x PBST), biotin-conjugated anti-mouse IgG and IgA antibodies (Southern Biotech) were added at a concentration of 0.5 μg/mL diluted in PBST 1% FCS and incubated overnight at 4°C. Plates were washed (4x PBST) before incubation with a 1:1000 dilution of HRP avidin D (Vector Laboratories) in supplemented PBS (1X PBS, 0.05% Tween 20, 1% FCS) for 1–3 hr at room temperature. After washes (3x PBST, 3x PBS), AEC substrate (0.3mg 3-amino-9-ethylcarbazole in 0.1 M Na-Acetate buffer, pH 5, 0.3% hydrogen peroxide) was added and color reactions were allowed to proceed for 2–10 minutes before washing with distilled water. Plates were kept in the dark to dry until read and counted with the aid of a CTL ImmunoSpot 5.1.36 analyzer.

Hudson L.E., McDermott C.D., Stewart T.P., Hudson W.H., Rios D., Fasken M.B., Corbett A.H, & Lamb T.J. (2016). Characterization of the Probiotic Yeast Saccharomyces boulardii in the Healthy Mucosal Immune System. PLoS ONE, 11(4), e0153351.

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ELISPOT Assay for Human IgG Detection

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Ninety-six well filter plates (Millipore–MSHAN4B50) were coated with either VZV cell lysate (Meridian Life Sciences) at 5 μg/well or Affinipure donkey anti-human IgG (H+L; Jackson Immunoresearch) at 100 ng/well in PBS overnight at 4°C. The plates were washed with PBS four times and complete FBS media added for 2 hr at 37°C. Freshly prepared PBMCs were resuspended in complete FBS media at 1×10⁷/ml and added to each well, followed by threefold dilution and then incubated overnight at 37°C. The next day, the plates were washed four times with PBS, four times with PBS-T and incubated for 1.5 hr at room temperature with Biotin-SP-Affinipure F(ab′)₂ donkey anti-human IgG (Jackson Immunoresearch) at 100 ng/well in PBS + 10% FCS + 0.05% Tween 20 (PBS-T-FBS). Plates were washed four times with PBS-T and incubated for 1.5 hr with HRP-avidin D (Vector laboratories) at 1:1000 in PBS-T-FBS. Plates were washed four times with PBS-T, four times with PBS and then developed using AEC substrate (3 amino-9 ethyl-carbazole; Sigma Aldrich). Developed plates were dried, scanned and analyzed using an automated ELISPOT counter (Cellular Technologies, Ltd).

Li S., Sullivan N.L., Rouphael N., Yu T., Banton S., Maddur M.S., McCausland M., Chiu C., Canniff J., Dubey S., Liu K., Tran V., Hagan T., Duraisingham S., Wieland A., Mehta A., Whitaker J.A., Subramaniam S., Jones D.P., Sette A., Vora K., Weinberg A., Mulligan M.J., Nakaya H.I., Levin M., Ahmed R, & Pulendran B. (2017). Metabolic Phenotypes Of Response to Vaccination in Humans. Cell, 169(5), 862-877.e17.

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BG505-specific ASCs Quantification by ELISPOT

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BG505-specific Ab-secreting cells (ASCs) were detected by Enzyme-Linked ImmunoSpot (ELISPOT) assay on fresh cells from blood, obtained 5 days after the second immunization. 96-well multi-screen HTS HA filter plates, opaque (Millipore) were coated with 20 μg/mL of unconjugated Galanthus nivalis lectin (Vector labs) to capture antigen. Plates were coated overnight, washed, and blocked with complete RPMI medium at 37°C. 20 μg/mL of BG505 SOSIP.664 protein (laboratory of J.P. Moore, Cornell University) was added and incubated 1 hr at 37°C. All washes for GNL-BG505 plates were performed with PBS only. Plates were washed, mononuclear cells were plated in 3-fold serial dilutions, and incubated overnight 37°C. Plates were washed and incubated for 2 hr at RT with anti-monkey biotin conjugated IgG (Rockland) diluted at 1/1,000 in PBS with 1% FBS solution. Plates were washed and HRP avidin D (Vector labs) was added at 1/1,000 dilution and incubated for 2 hr at RT. The last wash was performed with PBS-T followed by PBS. An AEC Substrate Set (BD Biosciences) was used to develop spots. Plates were scanned and counted at Zellnet Consulting using the Immunospot CTL image acquisition (Cellular Technologies). Spots are reported as antigen-specific ASCs per million mononuclear cells.

Pauthner M., Havenar-Daughton C., Sok D., Nkolola J.P., Bastidas R., Boopathy A.V., Carnathan D.G., Chandrashekar A., Cirelli K.M., Cottrell C.A., Eroshkin A.M., Guenaga J., Kaushik K., Kulp D.W., Liu J., McCoy L.E., Oom A.L., Ozorowski G., Post K.W., Sharma S.K., Steichen J.M., de Taeye S.W., Tokatlian T., Torrents de la Peña A., Butera S.T., LaBranche C.C., Montefiori D.C., Silvestri G., Wilson I.A., Irvine D.J., Sanders R.W., Schief W.R., Ward A.B., Wyatt R.T., Barouch D.H., Crotty S, & Burton D.R. (2017). Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches. Immunity, 46(6), 1073-1088.e6.

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Shrimp Allergen IgE Binding Assay

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Purified recombinant shrimp allergens diluted in coating buffer (100 mM Na₂CO₃, 100 mM NaHCO₃, pH 9.6) were coated onto MaxiSorp microtiter plates (Nunc, Carlsbad, CA, USA) and incubated overnight at 4°C. After washing the plates with 0.05% Tween‐20/PBS (PBS‐T) and blocking the plates with 5% foetal bovine serum (Gibco, Thermo Fisher Scientific) diluted in PBS (blocking buffer) at room temperature for 2 h, serum samples diluted at 1:10 in blocking buffer were added for overnight incubation at 4°C. IgE binding was detected by incubating the plates with biotinylated anti‐human IgE antibodies (1:1000 dilution, Vector Labs, Burlingame, CA, USA), HRP avidin D (1:1000 dilution, Vector Labs) and TMB substrate (BD Biosciences, Franklin Lakes, NJ, USA). Upon terminating the reaction with 0.1 M sulfuric acid, the optical density (OD) at 450 nm was measured using a microplate reader (BioTek, Santa Clara, CA, USA).²¹ Results were considered positive only at OD >2 fold ± SD over negative controls (sera of ten non‐atopic control subjects).

Wai C.Y., Leung N.Y., Leung A.S., Ngai S.M., Pacharn P., Yau Y.S., Rosa Duque J.S., Kwan M.Y., Jirapongsananuruk O., Chan W.H., Chua G.T., Lee Q.U., Piboonpocanun S., Ho P.K., Wong J.S., Li S., Xu K.J., Wong G.W., Chu K.H., Leung P.S., Vichyanond P, & Leung T.F. (2022). Comprehending the allergen repertoire of shrimp for precision molecular diagnosis of shrimp allergy. Allergy, 77(10), 3041-3051.

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Quantifying antigen-specific B cells

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PVDF plates (EMD Millipore) were activated with 35% EtOH for 5 min 22°C. After substantial washes with dH₂O and PBS, wells were coated overnight at 22°C in humid chamber with 0.75 µg of goat-anti-mouse-IgG (Jackson Laboratories) in PBS.
At d 3, 7, 9, 14 and 17 mediastinal lymph nodes were excised and individually processed in a single cell suspension. Plates were blocked with PBS plus 2% BSA for 1 h at 22°C and washed with PBST and RPMI. Cells were incubated in serial twofold dilutions starting at 5 × 10⁵ in RPMI plus 7% FCS for 18–20 h at 37 °C.
Cells were lysed with extensive dH₂O washes and wells incubated with 5 ng of rHA for 2 h at 22°C. Detection was carried out after 1 h incubation with Avidin D-HRP (VectorLabs) 1:1000 by 10 minutes incubation with AEC substrate set (BD Biosciences). Spots were counted for each well with CTL ImmunoSpot analyzer and data analyzed with CTL ImmunoSpot software.

Angeletti D., Gibbs J.S., Angel M., Kosik I., Hickman H.D., Frank G.M., Das S.R., Wheatley A.K., Prabhakaran M., Leggat D.J., McDermott A.B, & Yewdell J.W. (2017). Defining B Cell Immunodominance to Viruses. Nature immunology, 18(4), 456-463.

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Immunohistochemical Analysis of Parkinson's Markers

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Immunohistochemical staining for α-synuclein, glucocerebrosidase, DAT and TH was performed in serially sectioned, free-floating vibratome sections. The sections were incubated overnight at 4°C with a polyclonal antibody against total α-synuclein (1:500, affinity-purified rabbit polyclonal antibody; Millipore) (71 (link)), glucocerebrosidase (mouse monoclonal antibody; Abnova, Walnut, CA), TH (mouse monoclonal antibody; Millipore, Billerica, MA) or DAT (mouse monoclonal antibody; Millipore, Billerica, MA) followed by a secondary biotinylated antibody (1:100, Vector Laboratories, Inc., Burlingame, CA) and Avidin D-HRP (1:200, ABC Elite, Vector). Detection was performed with 3,3′-diaminobenzidine (72 (link)). A subset of sections that were immunostained with the α-synuclein antibody was subjected to proteinase K pre-treatment (8 min, 10 µg/ml). All sections were processed simultaneously using the same conditions. Immunostained slides were analyzed with a digital Olympus bright field digital microscope (BX41). For each animal, a total of three sections (four digital images per section at 400× magnification) were obtained from the striatum and analyzed as previously described with the ImageJ program (NIH) (71 (link)).

Rockenstein E., Clarke J., Viel C., Panarello N., Treleaven C.M., Kim C., Spencer B., Adame A., Park H., Dodge J.C., Cheng S.H., Shihabuddin L.S., Masliah E, & Sardi S.P. (2016). Glucocerebrosidase modulates cognitive and motor activities in murine models of Parkinson’s disease. Human Molecular Genetics, 25(13), 2645-2660.

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Quantifying antigen-specific B cells

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Enumeration of Antibody-Secreting Cells in Lymph Nodes

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Standard ELISPOT assays were performed as described previously [14 (link)]. Ninety six well plates (EMD Millipore, Billerica, MA, USA) were coated with 10 µg/mL of anti-monkey IgG, IgA and IgM (H&L) goat antibody (Rockland Immunochemicals, Pottstown, PA, USA) or 2 µg/mL of recombinant SIV ENV overnight at 4 °C for enumeration of antibody secreting cells (ASCs). Wells were blocked with complete RPMI medium for 2 h at 37 °C. Whole lymph node cell preparations were diluted, plated in serial 3-fold dilutions and incubated overnight at 37 °C. Wells were then incubated for 2h at RT with biotin-conjugated anti-monkey IgG (Rockland), diluted 1:1000 in PBS with 0.05% Tween 20 and 1% FBS. Wells were incubated for 3h at RT with Avidin D-HRP (Vector labs, Burlingame, CA, USA), diluted 1:1000. Spots were developed with filtered 3-amino-9-ethylcarbazole substrate and then counted using the Immunospot CTL counter and the Image Acquisition v4.5 software (Cellular Technology, Shaker Heights, OH, USA).

Hong J.J., Silveira E.L., Amancha P.K., Byrareddy S.N., Gumber S., Chang K.T., Ansari A.A, & Villinger F. (2017). Early initiation of antiretroviral treatment post SIV infection does not resolve lymphoid tissue activation. AIDS (London, England), 31(13), 1819-1824.

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Immunohistochemical Staining of GFAP

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Slides were incubated in rabbit anti-cow GFAP antibody (Dako, Glostrup, Denmark) at a dilution of 1:500 in 4% horse serum in DPBS overnight. Tissues were then washed 3 times in DPBS and incubated in biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) diluted at 1:10,000 in 4% horse serum in DPBS for 4 hours. Following secondary antibody incubation, Avidin D-HRP (Vector Laboratories, Burlingame, CA) diluted at 1:1,000 in DPBS for 1 hour was applied. DAB chromagen solution (Vector Laboratories, Burlingame, CA) was applied for 5 minutes. Tissues were then rinsed 3 times in DPBS and dried overnight.

Turner R.C., VanGilder R.L., Naser Z.J., Lucke-Wold B.P., Bailes J.E., Matsumoto R.R., Huber J.D, & Rosen C.L. (2014). Elucidating the Severity of Preclinical Traumatic Brain Injury Models: A Role for Functional Assessment?. Neurosurgery, 74(4), 382-394.

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