Elisa assay diluent

Manufactured by BioLegend

Sourced in United States, Latvia

ELISA Assay Diluent is a buffer solution designed for use in Enzyme-Linked Immunosorbent Assay (ELISA) procedures. Its core function is to dilute samples and standards, providing a consistent medium to ensure accurate and reliable ELISA results.

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Lab products found in correlation

9 protocols using elisa assay diluent

ELISA-based Silk Protein Quantification

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The gene-transfected DO-11.10 cells were lysed with RIPA buffer (50 mM Tris-HCL pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail; Nacalai Tesque) on ice for 1 h. Cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C and the supernatants used for ELISA. The stock silk solutions (80 mg/mL in 9 M LiBr) were diluted with 1 mM Tris-HCl (pH 8.0) to a concentration of 0.25 mg/mL. One hundred microlitres of the cell lysate, culture supernatants from hybridoma cells, and diluted silk solutions were applied to 96-well plates and incubated overnight at 4 °C. After three washes with PBS, each well was blocked with ELISA Assay Diluent (BioLegend, San Diego, CA, USA) at room temperature for 1 h. After five washes with PBS and Tween 20, CEA (Abcam, code no. ab742, Cambridge, UK) was added to the wells and incubated at room temperature for 2 h. Binding was detected with anti-CEA polyclonal antibody (Abcam, code no. ab15987), then HRP-conjugated anti-rabbit Igs (Dako), finally by incubation with ELISA POD Substrate TMB solution (Nacalai Tesque). After colour development, the reaction was stopped with 2 N H₂SO₄ and the absorbance read at 450 nm using a microplate reader (iMark^TM Microplate Reader; Bio-Rad).

Sato M., Kitani H, & Kojima K. (2017). Development and validation of scFv-conjugated affinity silk protein for specific detection of carcinoembryonic antigen. Scientific Reports, 7, 16077.

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Skin Protein Quantification and Cytokine Analysis

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A piece of back skin was snap frozen and stored at −80°C. Samples were homogenized with a Precellys homogenizer (Bertin) in Precellys tubes (Peqlab). Total protein quantification in skin lysates was done by Bradford protein assay according to the manufacturer´s protocol (Bio‐Rad). 40 (ELISA) or 100 µg (Luminex) of total protein diluted in ELISA assay diluent (BioLegend) with a protease inhibitor cocktail (Roche) were used to perform an ELISA for CCL2 (BD Biosciences) or a Multiplex Luminex assay (Thermo Fisher Scientific) according to manufacturer’s instructions. Analysis of the Multiplex Luminex assay was done on a Luminex MAGPIX System using the xPONENT Software.

Novoszel P., Holcmann M., Stulnig G., De Sa Fernandes C., Zyulina V., Borek I., Linder M., Bogusch A., Drobits B., Bauer T., Tam‐Amersdorfer C., Brunner P.M., Stary G., Bakiri L., Wagner E.F., Strobl H, & Sibilia M. (2021). Psoriatic skin inflammation is promoted by c‐Jun/AP‐1‐dependent CCL2 and IL‐23 expression in dendritic cells. EMBO Molecular Medicine, 13(4), e12409.

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Antibody Titer Determination for Tissue Injury

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PCL particles were dissolved in chloroform to coat the bottom of 96-well plates (20 mg/ml). Sera collected 3, 6, and 12 weeks after injury were serially diluted (in the range of 1:50 to 1:102,400) in ELISA Assay Diluent (BioLegend, catalog no. 421203). Before loading, the PCL-coated plate was blocked with the assay diluent for 1 hour.
After blocking, each dilution of the serum sample was loaded into the plate and incubated for 2 hours. After washing, biotin anti-mouse IgG1 or IgM or IgA was added to capture the bound antibody for 1 hour, followed by washing. Streptavidin solution was added to the wells and incubated for 30 min. Trimethylboron substrate was used for HRP detection and stopped with H₂SO₄. Absorbance was read at 450 and 570 nm (background control).

Chung L., Maestas DR J.r., Lebid A., Mageau A., Rosson G.D., Wu X., Wolf M.T., Tam A.J., Vanderzee I., Wang X., Andorko J.I., Zhang H., Narain R., Sadtler K., Fan H., Čiháková D., Le Saux C.J., Housseau F., Pardoll D.M, & Elisseeff J.H. (2020). Interleukin 17 and senescent cells regulate the foreign body response to synthetic material implants in mice and humans. Science translational medicine, 12(539), eaax3799.

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Detection of Topoisomerase-DNA Adducts

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Primary and secondary antibodies were diluted in 1× ELISA Assay Diluent (Biolegend). Top1–DNA adducts were detected with polyclonal rabbit anti-human Top1 IgG (ab28432, Abcam) at 1:2000 dilution. Top2a–DNA adducts were detected with monoclonal mouse anti-human Top2a IgG (#611326, BD Transduction Laboratories) at 1:500. Gyrase–DNA adducts were detected with polyclonal rabbit anti-GyrA antibodies (PA001, Inspiralis) at 1:500 dilution. Secondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG at 1:5000 or 1:2500, respectively.

Kiianitsa K, & Maizels N. (2014). Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay. Nucleic Acids Research, 42(13), e108.

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TCR Cloning and Antigen Specificity Assay

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The TCR cloning and expression protocol^{10 (link)} is described in the Supplementary Information. We used TCR-deficient Jurkat cells (JurkatΔαβ)^{35 (link)} transduced to express CD4 and CD8 as reporter cells. To assess antigen specificity of the cloned TCRs, we pulsed dendritic cells derived from patient PBMCs with candidate peptides (10 μg ml⁻¹ unless specified otherwise) for 2 h in complete RPMI. Reporter cells stably expressing TCR were co-cultured in 96-well round bottom plate with pulsed dendritic cells, in a ratio of 20:1 (0.5 × 10⁶ Jurkat-expressing TCR cells to 2.5 × 10⁴ dendritic cells) in complete RPMI. After 24 h at 37 °C and 5% CO_2, the supernatant was collected and diluted 1:2 with ELISA Assay Diluent (Biolegend) and IL-2 production was measured using the Human IL-2 ELISA MAX Standard Kit (Biolegend) according to the manufacturer’s instructions. OVA peptide was used as a negative control and PMA/ionomycin was used as a positive control.

Keskin D.B., Anandappa A.J., Sun J., Tirosh I., Mathewson N.D., Li S., Oliveira G., Giobbie-Hurder A., Felt K., Gjini E., Shukla S.A., Hu Z., Li L., Le P.M., Allesøe R.L., Richman A.R., Kowalczyk M.S., Abdelrahman S., Geduldig J.E., Charbonneau S., Pelton K., Iorgulescu J.B., Elagina L., Zhang W., Olive O., McCluskey C., Olsen L.R., Stevens J., Lane W.J., Salazar A.M., Daley H., Wen P.Y., Chiocca E.A., Harden M., Lennon N.J., Gabriel S., Getz G., Lander E.S., Regev A., Ritz J., Neuberg D., Rodig S.J., Ligon K.L., Suvà M.L., Wucherpfennig K.W., Hacohen N., Fritsch E.F., Livak K.J., Ott P.A., Wu C.J, & Reardon D.A. (2018). Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial. Nature, 565(7738), 234-239.

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Quantification of Allergen-specific IgE Antibodies

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Allergen-specific IgE antibody determination was performed as previously described (11 (link)). Briefly, 96-well microplates were immobilized with 5 µg/10 mL (plate) of recombinant Art v 1 protein on an ELISA Coating Buffer (BioLegend) overnight. On the following day, ELISA Assay Diluent (BioLegend) in phosphate-buffered saline (PBS) was added into the plates at 200 μL/well and incubated on a PST-60HL thermal shaker (BIOSAN, Latvia) for 1 h at room temperature (RT). The plates were then washed four times with ELISA Wash Buffer (BioLegend). The mouse serum samples were diluted in a 1:5 ratio with ELISA Assay Diluent and 100 µl were added to the wells and incubated for 1.5-2 h at RT with stirring. After washing (4x), anti-mouse biotinylated detection antibodies for IgE (1:200, Cat # 406904, BioLegend, 100 µl/well) were added and the plates were incubated for 1 h at RT with stirring. After additional washing (4x), plates were incubated with HRP Streptavidin (BioLegend, 1:1000, 100 µL/well) for 30 min at RT with stirring. After that, the washing (5x) was repeated, and on completion of which the TMB substrate (BioLegend, 100 µl/well) was added. The color reaction was stopped by adding a stop solution (100 µL/well), and the optical density was measured at 450 nm on a Stat Fax 2100 analyzer (Awareness Tech).

Babayeva M., Tabynov K., Nurpeisov T., Fomin G., Renukaradhya G.J., Petrovsky N, & Tabynov K. (2022). A recombinant Artemisia vulgaris pollen adjuvanted Art v 1 protein-based vaccine treats allergic rhinitis and bronchial asthma using pre- and co-seasonal ultrashort immunotherapy regimens in sensitized mice. Frontiers in Immunology, 13, 983621.

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Quantification of CCL2 and IL-17A

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100 μg of total skin protein extracts prepared in RIPA lysis buffer was diluted in ELISA assay diluent (BioLegend) with Complete protease inhibitor cocktail (Roche), and ELISA was performed for CCL2 and IL‐17A (BD Biosciences) according to the manufacturer's instructions. Absorbance was measured with a Tecan plate reader (Tecan Infinite 200 Pro fluorometer).

Malik B., Vokic I., Mohr T., Poppelaars M., Holcmann M., Novoszel P., Timelthaler G., Lendl T., Krauss D., Elling U., Mildner M., Penninger J.M., Petzelbauer P., Sibilia M, & Csiszar A. (2023). FAM3C/ILEI protein is elevated in psoriatic lesions and triggers psoriasiform hyperproliferation in mice. EMBO Molecular Medicine, 15(7), e16758.

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Cytokine Profiling in Immune Cell Cultures

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Cell culture supernatants were collected from (1) BMDC (100,000 cells/well) cultures 8 hours after stimulation, (2) DC-T cocultures after a 3-day incubation period, or (3) total draining lymph node (dLN) cell cultures (500,000 cells/well) kept in culture for 2 days, and stored frozen at −80°C in single-use aliquots.
For sandwich Enzyme-Linked Immunosorbent Assay (ELISA), cell culture supernatants were diluted using the ELISA assay diluent (Biolegend 4212013) and cytokine concentrations were measured using the ELISA MAX standard set mouse IFN-γ (BioLegend 430801) and IL-17A (BioLegend 432501) kits according to the manufacturer’s instructions.
For flow cytometric, bead-based immunoassays, DC and dLN cell culture supernatants were diluted and processed using the LEGENDplex mouse anti-virus response panel (BioLegend 740622) and the LEGENDplex mouse Th cytokine panel (BioLegend 740740) kits, respectively. Data were acquired on the NovoCyte flow cytometer (Acea Biosciences/Agilent) and analyzed using the LEGENDplex software v8 (BioLegend).

Pandey S., Gruenbaum A., Kanashova T., Mertins P., Cluzel P, & Chevrier N. (2020). Pairwise Stimulations of Pathogen-Sensing Pathways Predict Immune Responses to Multi-Adjuvant Combinations. Cell systems, 11(5), 495-508.e10.

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ELISA for Evaluating β2-m Binding

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ELISA was used for evaluating the β2-m-binding activity of ApoE NTD-MHC α3 and ApoE NTD-scFv, as described previously, 2 with minor modifications. Briefly, ApoE NTD-MHC α3/scFv samples in PBS at different concentrations (0, 50, 100, 150, 200, 300, 400, and 500 nM) were coated onto a 96-well assay polystyrene plate (AGC Techno Glass, Japan), which was then washed five times with PBS containing 0.05% (v/v) Tween 20 (PBS-T). After blocking with a blocking buffer (ELISA assay diluent; BioLegend, CA), β2-m (0 or 1 µg mL -1 ) in the blocking buffer or in fetal bovine serum (FBS) (Equitech-Bio, TX) was added to the protein-coated plate. After incubation and washing, rabbit anti-human β2-m PAb (13511-1-AP; Proteintech, IL), followed by HRP-conjugated swine anti-rabbit Ig PAb (P0399; Dako, Denmark), was added to the plate. The plate was then washed with PBS-T, and β2-m bound to the protein-coated plate was detected using ELISA POD substrate TMB solution (Nacalai Tesque, Japan). Absorbance at 450 nm was measured using a plate reader (Varioskan™; Thermo Fisher Scientific, MA). Background (0 µg mL -1 β2-m) absorbance was subtracted from the sample (1 µg mL -1 β2-m) absorbance. Three different wells were used for each condition (n = 3).

Kambe Y., Kuwahara K., Sato M., Nakaoki T, & Yamaoka T. (2021). Enhanced β2-microglobulin binding of a "navigator" molecule bearing a single-chain variable fragment antibody for artificial switching of metabolic processing pathways. Biomaterials science, 9(16).

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