Alphalisa immunoassay kit

Manufactured by PerkinElmer

Sourced in United States

The AlphaLISA immunoassay kit is a homogeneous bead-based assay technology developed by PerkinElmer. It is designed to detect and quantify analytes in biological samples, such as proteins, peptides, and small molecules. The assay relies on the formation of a complex between the analyte and specific antibodies or binding proteins, which triggers a luminescent signal that can be measured using a compatible microplate reader.

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Lab products found in correlation

Envision plate reader, by PerkinElmer (1 mentions) Sea peptide, by Toxin Technology (1 mentions) Ensight instrument, by PerkinElmer (1 mentions) G 5009, by Merck Group (1 mentions) Pherastar fs plate reader, by BMG Labtech (1 mentions) Serum triglyceride determination kit, by Merck Group (1 mentions)

Spelling variants of this product

AlphaLISA kit (37 citations) AlphaLISAs (2 citations) AlphaLISA IL-6 Immunoassay Research kit (2 citations) AlphaLISA immunoassay detection kit (2 citations) AlphaLISA Human Insulin Immunoassay kit (2 citations)

9 protocols using alphalisa immunoassay kit

IL-8 Quantification via AlphaLISA

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IL-8 was quantified using an AlphaLISA immunoassay kit (AL224C, Perkin-Elmer, Waltham, MA, USA) according to the manufacturer’s recommendations.

Cadau S., Gault M., Berthelemy N., Hsu C.Y., Danoux L., Pelletier N., Goudounèche D., Pons C., Leprince C., André-Frei V., Simon M, & Pain S. (2022). An Inflamed and Infected Reconstructed Human Epidermis to Study Atopic Dermatitis and Skin Care Ingredients. International Journal of Molecular Sciences, 23(21), 12880.

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HA Production in Hyalocytes and A549 Cells

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To perform the HA assay, 5,000 hyalocytes/well were seeded in 48-well plates containing 0.5 ml IMDM (+ 10% FBS) and stimulated for 6 days with different agents: 10 ng/ml of bFGF, 5 ng/ml of TGF-β, 10 ng/ml of PDGF-BB, 200 µg/ml of AsA, 1 mg/ml of DEX, and 5 µM of H₂O₂. HA levels were detected in 10 µl of primary culture supernatant using the hyaluronic acid (human) AlphaLISA^® immunoassay kit (PerkinElmer, Waltham, MA). Two different types of cells, lung adenocarcinoma (A549) cells (ATCC, LGC Standards S.r.l., Milan, Italy), were used for comparison purposes based on their different HA production in 6 days [21 (link),22 (link)]. HA levels were measured with an EnVision plate reader (PerkinElmer), and the values are expressed as ng HA/ng cell protein.

Nuzzi R., Bergandi L., Zabetta L.C., D’Errico L., Riscaldino F., Menegon S, & Silvagno F. (2020). In vitro generation of primary cultures of human hyalocytes. Molecular Vision, 26, 818-829.

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Investigating T Cell Activation Modulation

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Human blood was purchased from Research Blood Components (Boston, MA). Human PBMC were isolated by density centrifugation and stimulated with SEA peptide (Toxin Technology), which mediates interactions between MHC class II molecules and CD86 on APCs and the TCR and CD28 on T cells, respectively [33 (link), 34 (link)]. AGEN1884 or AGEN2041 were added alone or in combination with anti-PD-1 (nivolumab, pembrolizumab or AGEN2034), LAG-3 (clone 25F7), CD137 (clone 10C7) or an isotype control antibody (clone 4Ab-028). After 5 days, co-culture supernatants were collected and interleukin-2 (IL-2) was measured as an endpoint of enhanced T cell responsiveness using an AlphaLISA® immunoassay kit (Perkin-Elmer), as per manufacturer’s instructions.

Gombos R.B., Gonzalez A., Manrique M., Chand D., Savitsky D., Morin B., Breous-Nystrom E., Dupont C., Ward R.A., Mundt C., Duckless B., Tang H., Findeis M.A., Schuster A., Waight J.D., Underwood D., Clarke C., Ritter G., Merghoub T., Schaer D., Wolchok J.D., van Dijk M., Buell J.S., Cuillerot J.M., Stein R., Drouin E.E, & Wilson N.S. (2018). Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody. PLoS ONE, 13(4), e0191926.

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Quantifying Vitreous Hyaluronic Acid

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HA assay was performed in 20 μl of vitreous samples using the human hyaluronic acid AlphaLISA® immunoassay kit according to the manufacturer's instructions (PerkinElmer, Waltham, Massachusetts, United States). The assay shows no cross-reactivity with human collagen Type I, Type II, Type III, and fibronectin. Absorbance was measured at 615 nm with the EnSight instrument (PerkinElmer, Waltham, Massachusetts, United States). Kaleido version 2.0.3058.126 and GraphPad Prism (5.01) software were used for data acquisition and elaboration. HA levels were expressed as ng HA/μl of vitreous samples.

Bergandi L., Skorokhod O.A., La Grotta R., Schwarzer E, & Nuzzi R. (2019). Oxidative Stress, Lipid Peroxidation, and Loss of Hyaluronic Acid in the Human Vitreous Affected by Synchysis Scintillans. Journal of Ophthalmology, 2019, 7231015.

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Insulin quantification by TPX and AlphaLISA

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Insulin was analyzed using either the ArcDia 2-photon fluorescence excitation microparticle fluorometry (TPX) assay (ArcDia Group, Turku, Finland) [29 (link)], or the AlphaLISA immunoassay kit (Perkin Elmer, Waltham, MA). The assays were performed according to the manufacturer's instructions, using a recombinant human insulin standard to determine the insulin concentration.

Rajasekaran S.S., Kim J., Gaboardi G.C., Gromada J., Shears S.B., dos Santos K.T., Nolasco E.L., Ferreira S.D., Illies C., Köhler M., Gu C., Ryu S.H., Martins J.O., Darè E., Barker C.J, & Berggren P.O. (2018). Inositol hexakisphosphate kinase 1 is a metabolic sensor in pancreatic β-cells. Cellular Signalling, 46, 120-128.

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Cytokine Profiling in Transwell Co-Culture

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Cytokine titres were determined using an AlphaLISA Immunoassay kit (Perkin Elmer) according to the manufacturer’s instructions. The sum of cytokines detected in the upper and lower compartments of the transwell is shown for each of the monocultured samples, while cytokine levels were kept separated for the upper and lower compartment for the co‐cultured samples.

Schimmel L., Chew K.Y., Stocks C.J., Yordanov T.E., Essebier P., Kulasinghe A., Monkman J., dos Santos Miggiolaro A.F., Cooper C., de Noronha L., Schroder K., Lagendijk A.K., Labzin L.I., Short K.R, & Gordon E.J. (2021). Endothelial cells are not productively infected by SARS‐CoV‐2. Clinical & Translational Immunology, 10(10), e1350.

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Insulin Secretion Assay in INS-1 Cells

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INS-1 832/13 cells or INS-1 832/13 stably expressing GFP-hSurf4 cells were transfected twice with RNAi oligonucleotides as described above and treated with modified Krebs–Ringer buffer (KRB; containing 120 mM NaCl, 24 mM NaHCO₃, 1 mM MgCl₂, 2 mM CaCl₂, and 15 mM HEPES at pH 7.4 and supplemented with 0.1% BSA and 2.8 mM glucose) for 2 h, followed by incubation with the same KRB buffer (low glucose) or that containing 25 mM glucose (high glucose) for 2 h. The amount of secreted insulin was measured using an AlphaLISA immunoassay kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions.

Saegusa K., Matsunaga K., Maeda M., Saito K., Izumi T, & Sato K. (2022). Cargo receptor Surf4 regulates endoplasmic reticulum export of proinsulin in pancreatic β-cells. Communications Biology, 5, 458.

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AlphaLISA Immunoassay for Cytokine Quantification

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Human CXCL10 (AL259C), human CXCL9 (AL280C), human IL-8 (AL224C) and human IL-6 (AL223C) AlphaLISA immunoassay kits were purchased from Perkin Elmer and performed according to the manufacturer's protocol. For cell culture supernatant samples, the assay buffer contained 25 μM HEPES, 50 mM NaCl, 10 mM sodium EDTA, 2 mg/mL dextran (Mw. 500,000), 0.5% casein and 0.05% Tween-20, adjusted to pH 7.4. For plasma samples, the assay buffer also contained 0.1% bovine gamma globulin (G-5009, Sigma Aldrich) and 0.2 μg/mL HBR1 (3KC533, Scantibodies). Standards, biotinylated detection antibodies, and acceptor bead solutions were prepared in assay buffer. The assays were performed in 384-well white polypropylene conical-bottom microplates (4307, Thermo Scientific). Two microliters of standards, cell culture supernatant or plasma sample were incubated with 10 μg/mL acceptor beads and 1 nM biotinylated detection antibody for 1 h at room temperature. Streptavidin-coated donor beads (40 μg/mL) were added to the mixture and incubated for an additional 30 min. The plate was read on a PHERAstar FS plate reader (BMG LabTech) where the bead complex was excited at 680 nm for 1 s and emission signals were collected at 615 nm for 1 s after a 40-millisecond delay.

Simon A.B., Frampton J.P., Huang N.T., Kurabayashi K., Paczesny S, & Takayama S. (2014). Aqueous two-phase systems enable multiplexing of homogeneous immunoassays. Technology, 2(2), 176-.

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Plasma Metabolic Biomarkers Measurement

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Plasma triglycerides (TG) and glycerol were measured using a Serum Triglyceride Determination Kit (Sigma, Oakville, Canada) following the manufacturer's protocol. Plasma insulin, C-peptide, adiponectin and leptin levels were measured using bead-based AlphaLISA immunoassay kits (Perkin Elmer, Waltham, MA) according to the manufacturer's instructions. Plasma renin activity was calculated by measuring Angiotensin I production by immunoassay kit (Diagnostic Biochem Canada, Dorchester, ON, Canada).

Shamansurova Z., Tan P., Ahmed B., Pepin E., Seda O, & Lavoie J.L. (2016). Adipose tissue (P)RR regulates insulin sensitivity, fat mass and body weight. Molecular Metabolism, 5(10), 959-969.

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