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High sensitivity elisa kit

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The high sensitivity ELISA kit is a laboratory assay used for the quantitative measurement of specific analytes in biological samples. It employs the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the target analyte with high sensitivity.

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High sensitivity ELISA ELISAs

22 protocols using high sensitivity elisa kit

1

Cytokine Quantification in Conditioned Media

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Conditioned media (CM) were prepared by incubating cells for 18 h in serum-free medium after the end of treatments. Cells were counted for normalization purposes. After centrifugation, CM samples were stored at −20 °C until use. IL-6 was measured using high-sensitivity ELISA kit (Invitrogen), while IL-8 and MCP-1 with the multi-analyte ELISArray Handbook (Qiagen).
Prattichizzo F., De Nigris V., Mancuso E., Spiga R., Giuliani A., Matacchione G., Lazzarini R., Marcheselli F., Recchioni R., Testa R., La Sala L., Rippo M.R., Procopio A.D., Olivieri F, & Ceriello A. (2017). Short-term sustained hyperglycaemia fosters an archetypal senescence-associated secretory phenotype in endothelial cells and macrophages. Redox Biology, 15, 170-181.
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2

Cytokine Measurement from Cell Supernatants

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Culture supernatants were collected at the end of each incubation, centrifuged, and stored at −20°C until use in the assays. IL-6 and TNF-α concentrations were measured using a commercially available, high-sensitivity ELISA kit (Invitrogen) or a 4 custom cytokine multiplex (Tema Ricerca, Castenaso, Bologna).
Prattichizzo F., Giuliani A., Recchioni R., Bonafè M., Marcheselli F., De Carolis S., Campanati A., Giuliodori K., Rippo M.R., Brugè F., Tiano L., Micucci C., Ceriello A., Offidani A., Procopio A.D, & Olivieri F. (2016). Anti-TNF-α treatment modulates SASP and SASP-related microRNAs in endothelial cells and in circulating angiogenic cells. Oncotarget, 7(11), 11945-11958.
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3

Plasma Cytokine Profiling in Mice

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Blood was collected via cardiac puncture into tubes containing EDTA. Plasma was obtained by centrifugation at 1000g for 10 min and stored at −70 °C until analysis. Concentrations of IL-2, IL-4, IL-10 and IFN-γ in plasma were measured by enzyme-linked immunosorbent assay (ELISA) using High Sensitivity ELISA Kit (eBiosciences). IL-12p70 and IL-18 were quantified using mouse IL-12p70 Quantikine Reagent Kit (R & D Systems, Minneapolis, USA) and IL-18 Platinum ELISA Kit (eBiosciences) respectively.
Li L., Wang W., Pan H., Ma G., Shi X., Xie H., Liu X., Ding Q., Zhou W, & Wang S. (2017). Microwave ablation combined with OK-432 induces Th1-type response and specific antitumor immunity in a murine model of breast cancer. Journal of Translational Medicine, 15, 23.
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4

Quantification of Inflammatory Cytokines

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Cytokine measurements in the various figures were performed at different time intervals, which are indicated in the legends for the respective figures.
Concentrations of mouse TNF and IL-6 produced by bone marrow-derived cultured CD49b+ cells were measured using the ProcartaPlex Multiplex Immunoassay (Essential Th1/Th2, 6plex, catalog number EPX060–20831-901, Fisher Scientific) with detection limits of 0.39 pg/ml and 0.21 pg/ml, respectively. Concentrations of TNF produced by basophils isolated from the bone marrow were measured with a high sensitivity ELISA kit from eBioscience (catalog number BMS607HS) with a detection limit of 0.75 pg/ml.
Concentrations of TNF, IL-6, KC, and MIP-2 (for serum and intraperitoneal lavage fluids) were measured with ELISAs. The TNF and IL-6 ELISAs were from BioLegend (catalog numbers 430904 and 431304, respectively); and the KC and MIP-2 ELISAs were from Peprotech (catalog numbers 900-K127 and 900-K152, respectively). The detection limits for these assays were 4 pg/ml for TNF, 2 pg/ml for IL-6, 4 pg/ml for KC and 16 pg/ml for MIP-2.
Piliponsky A.M., Shubin N.J., Lahiri A.K., Truong P., Clauson M., Niino K., Tsuha A.L., Nedospasov S.A., Karasuyama H., Reber L.L., Tsai M., Mukai K, & Galli S.J. (2019). Basophil-derived tumor necrosis factor can enhance survival in a sepsis model in mice. Nature immunology, 20(2), 129-140.
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5

Quantification of Inflammatory Markers

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Serum levels of IL-1β and TNF-α were determined using a high sensitivity ELISA kit (catalogue no. 50–112-9749 for IL-1β) and (catalogue no. 50–112-8954 for TNF-α) from eBioscience (Fisher Scientific, Waltham, MA, USA), according to manufacturer instructions and as described previously by our group [25 (link)]. Inflammatory mediators were expressed as pg/ml and normalised to protein concentration in each sample.
Elshaer S.L., Alwhaibi A., Mohamed R., Lemtalsi T., Coucha M., Longo F.M, & El-Remessy A.B. (2019). Modulation of the p75 neurotrophin receptor using LM11A-31 prevents diabetes-induced retinal vascular permeability in mice via inhibition of inflammation and the RhoA kinase pathway. Diabetologia, 62(8), 1488-1500.
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6

Retinal TNFα Quantification Protocol

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Retinas were homogenized in 20 mM Tris-HCl pH 7.4, 10 mM EDTA containing protease inhibitor cocktail (Complete Protease Inhibitor Cocktail; Roche, Basel, Switzerland) and 200 μM phenylmethylsufloxifluoride (PMSF). The TNFα protein levels were estimated with a high sensitivity ELISA kit (eBioscience, Ireland, UK), according to the manufacturers’ instructions. Tissue TNFα levels were expressed as pg/mg protein.
Martínez-Fernández de la Cámara C., Hernández-Pinto A.M., Olivares-González L., Cuevas-Martín C., Sánchez-Aragó M., Hervás D., Salom D., Cuezva J.M., de la Rosa E.J., Millán J.M, & Rodrigo R. (2015). Adalimumab Reduces Photoreceptor Cell Death in A Mouse Model of Retinal Degeneration. Scientific Reports, 5, 11764.
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7

Autoimmune Biomarkers in Rheumatic Diseases

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All samples were stored at − 80 °C until use. Serum levels of C-reactive protein (CRP) were determined by an immuno-turbidimetric technique using an Olympus AU 400 biochemical analyzer (Olympus Optical, Tokyo, Japan), and erythrocyte sedimentation rate (ESR) was measured according to the Fahreus and Westergren method. ANAs were detected using indirect immunofluorescence on HEP2 cells, and the autoantibodies of the ENA complex (anti-U1RNP, anti-Ro, anti-La, anti-DNA-topoisomerase I, anti-Jo-1, anti-P protein, anti-Sm, and anti-centromere) were assayed by immunoblot. Plasma levels of Hsp90 were assessed by a high-sensitivity ELISA kit (eBioscience, Vienna, Austria) according to the manufacturer's protocol. The assay recognizes human Hsp90 alpha. The calculated sensitivity is 0.03 ng/mL. The absorbance value was established at 450 nm by an ELISA reader (SUNRISE; Tecan, Grödig, Austria).
Štorkánová H., Oreská S., Špiritović M., Heřmánková B., Bubová K., Komarc M., Pavelka K., Vencovský J., Distler J.H., Šenolt L., Bečvář R, & Tomčík M. (2021). Plasma Hsp90 levels in patients with systemic sclerosis and relation to lung and skin involvement: a cross-sectional and longitudinal study. Scientific Reports, 11, 1.
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8

Assessing Bacteremia and Inflammation in Sepsis

Cited 1 time
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Bacteremia was regularly assessed by culture of small blood samples taken from the tail vein as previously described (Steele et al., 2017 (link)). In another experiment, systemic inflammation was assessed by measuring IL-6 in plasma samples obtained from animals sacrificed on day 4 and 2 weeks alongside non-sepsis controls using a high sensitivity ELISA kit (eBIOSCIENCE, Vienna, Austria). TNFα and IL-10 were assessed in non-sepsis controls and animals with sepsis on day 4 and 2 weeks (in the same mice) using Meso Scale Discovery (Rockville, Maryland) customized V-PLEX multiplex assay. These assays were conducted in singlet due to limited sample volumes.
Owen A.M., Patel S.P., Smith J.D., Balasuriya B.K., Mori S.F., Hawk G.S., Stromberg A.J., Kuriyama N., Kaneki M., Rabchevsky A.G., Butterfield T.A., Esser K.A., Peterson C.A., Starr M.E, & Saito H. (2019). Chronic muscle weakness and mitochondrial dysfunction in the absence of sustained atrophy in a preclinical sepsis model. eLife, 8, e49920.
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9

Plasma Biomarkers in Clinical Cohort

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Plasma IL-6 concentrations were analyzed using a commercially available high sensitivity ELISA kit (eBioscience), according to the manufacturer's instructions. The limit of detection was 0.007 pg/ml. The intra- and inter-assay coefficients of variability for plasma IL-6 measurements were 10 and 12%, respectively. Plasma hsCRP concentrations were analyzed using a commercially available Instant ELISA kit (eBioscience), according to the manufacturer's instructions. The limit of detection was 3 pg/ml. The intra- and inter-assay coefficients of variability for plasma hsCRP measurements were 6 and 8%, respectively. WBC counts were obtained from a standard clinical complete blood count panel using a Sysmex XN 1000 (Sysmex).
de Punder K., Entringer S., Heim C., Deuter C.E., Otte C., Wingenfeld K, & Kuehl L.K. (2018). Inflammatory Measures in Depressed Patients With and Without a History of Adverse Childhood Experiences. Frontiers in Psychiatry, 9, 610.
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10

Cytokine Profiling in Tumor Tissue

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Blood was collected via the tail vein into tubes containing EDTA. Plasma was obtained by centrifugation at 1000 g for 10 min and stored at −70℃ until analysis. Concentrations of interleukin (IL)-2, IL-4, IL-10, IL-12, IL-18, and interferon (IFN)-γ in plasma were measured by ELISA using High Sensitivity ELISA Kit (eBiosciences).
Tumors in all groups were collected and stored in RNA Later (Servicebio, CFX) at −20℃. Total RNA was extracted from tumors using Qiagen RNeasy Mini Kit (Qiagen, Germany) and complementary DNA (cDNA) was synthesized using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, California, USA). Subsequently, cDNA was used to measure the messenger RNA level of IL-2, IL-4, IL-18, tumor necrosis factor (TNF)-α, IFN-γ, IL-10 by quantitative real-time PCR (qRT-PCR) using the ABI Viia7 RT-PCR System (Applied Biosystems). All the sequences of primers are listed in online supplemental table S1. A housekeeping gene GAPDH was used as an internal control. The relative quantification was performed with the comparative threshold cycle method as described by the manufacturer.
Kan X., Zhou G., Zhang F., Ji H., Shin D.S., Monsky W., Zheng C, & Yang X. (2022). Enhanced efficacy of direct immunochemotherapy for hepatic cancer with image-guided intratumoral radiofrequency hyperthermia. Journal for Immunotherapy of Cancer, 10(11), e005619.
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