On day 29, blood samples were obtained from the tail vein and retro-orbital plexus. Blood was incubated at room temperature for 20 minutes, then centrifuged, and serum was separated from the clot. The level of cortisol hormone was measured with standard clinical chemistry analysis on a Roche Cobase411 analyzer. The level of TNF – α protein in the sera was measured with a commercial ELISA Kit (MABTECH AB, Sweden) according to the manufacturer’s instructions.
Elisa kit
Manufactured by Mabtech
Sourced in Sweden, United States, Italy
The ELISA kit is a laboratory tool used to detect and quantify specific proteins or other molecules in a sample. It is a widely used analytical technique in various fields, including immunology, microbiology, and biochemistry. The kit contains the necessary reagents and materials to perform the Enzyme-Linked Immunosorbent Assay (ELISA) process, which involves the use of antibodies and color changes to identify and measure the target analyte.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Phosphate substrate, by Merck Group (2 mentions)
Softmax pro 5.2 rev c, by Molecular Devices (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Cobas e411 analyzer, by Roche (1 mentions)
Human ifn α, by Mabtech (1 mentions)
Dtx 880 multimode detector, by Beckman Coulter (1 mentions)
Lentiguide puro vector, by Addgene (1 mentions)
Bovine ifn γ elisa development kit, by Mabtech (1 mentions)
96 well plate, by Corning (1 mentions)
Anti human cd3 antibody clone okt3, by BioLegend (1 mentions)
Bs 800, by Mindray (1 mentions)
Rpmi 1640 glutamax medium, by Thermo Fisher Scientific (1 mentions)
Sea seb, by Merck Group (1 mentions)
Staphylococcal enterotoxin a and b, by Merck Group (1 mentions)
Phytohaemagglutinin pha, by Merck Group (1 mentions)
Angptl3, by R&D Systems (1 mentions)
One glo reagent, by Promega (1 mentions)
Cc220, by Bio-Rad (1 mentions)
65 protocols using elisa kit
1
Serum Cortisol and TNF-α Analysis
2
IFN-α Levels Quantification by ELISA
3
Cytokine Profiling in Leptospirosis
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Five ELISA assays were used to quantitate the levels of cytokines IL-10, IL-17A, IL-21, IL-23, and TNF-α levels in the serum of patients with leptospirosis (n=57) and a healthy group (n=12) of individuals. The ELISAs were carried out following the manufacturer's instructions of each ELISA kit (Mabtech, Sweden). Mean absorbance values were calculated, and data were analyzed using Graphpad Prism version 6.05 (Graphpad Software Inc) to determine the level of cytokines.
4
CRISPR Knockdown of KPTN and RGS19
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To knock down KPTN and RGS19, two sgRNAs targeting shared exons of all transcription isoforms were delivered by lentivirus into a Cas9-expression huh7, a human hepatoma cell line. Exon 4 of KPTN and exon 5 of RGS19 were targeted by a dual CRISPR strategy to create a 40 bp and 130 bp frame shift deletion, respectively. SgRNAs were carried by Lenti-Guide-Puro vector (addgene, #52963) and infected cells were treated with 10 ug/ml puromycin treatment for 3 days to eliminate the negative cell. Positive targeted cells were expanded in culture and passaged for assays. Cells for measurement of secretive triglycerides, cholesterol, and APOB100 were cultured for 16 h in serum-free medium. Medium triglycerides and cholesterol were enriched for five times by vacuum centrifuge and measured with colorimetric kits, triglyceride (cobas), and CHOL2 (cobas), respectively. The amount of medium APOB100 was measured with an ELISA kit (MABTECH).
5
Bovine IFN-γ Levels in Mycobacterium bovis Infection
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To investigate IFN-γ levels in peripheral blood of infected cattle, IFN-γ concentration in plasma were measured using Bovine IFN-γ ELISA development kit (Mabtech, Nacka Strand, Sweden). To investigate M. bovis-specific IFN-γ response in infected cattle, PBMCs (4 × 105 cells) from infected cattle were incubated with 1.5 μg/ml of heat-killed M. bovis in 96-well plates (Corning, Inc.) at 37°C under 5% CO2 for 5 days. Subsequently, culture supernatants were obtained from individual wells and were tested for bovine IFN-γ using the ELISA kit (Mabtech) as described above.
6
Viral Peptide-specific T-cell Stimulation
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Based on the signal intensity of the peptide microarray analysis and statistical significance, the top five viral peptides (regardless of pathogen) recognized by each TIB IgG sample were selected for chemical synthesis (Peptides and Elephants, Henningsdorf, Germany). Next, 1.0 × 105 TILs were cultured in 200 μl of T-cell medium in the presence of 5 μg/ml of the individual viral peptides in round-bottom 96-well tissue culture plates. Cells in T-cell medium alone served as negative control while cells incubated with 30 ng/mL of the anti-human CD3 antibody (clone OKT3, BioLegend, San Diego, CA) were used as positive control to induce maximal stimulation of the TCR. The cells were incubated for three days at 37 °C with 5% CO2, following which culture supernatants were harvested for measuring IFN-γ production using a standard sandwich enzyme-linked immunosorbent assay (ELISA) kit (Mabtech, Stockholm, Sweden). The peptide-specific IFN-γ production is reported after subtracting values from the negative control (T-cell medium only) to reflect the readout as IFN-γ production (in pg/3 days/1.0 × 105 T cells).
7
Measuring Total IgG in Maternal and Cord Plasma
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Total IgG in maternal peripheral and cord plasma was measured at 1:400 000 dilution using a total IgG enzyme-linked immunosorbent assay (ELISA) kit (MabTech, Cincinnati, OH) and standards from the National Institute for Biological Standards and Controls. Hypergammaglobulinemia was defined as having total IgG >1600 mg/dL [13 (link), 34 (link), 35 (link)]. Optical density values were converted to total IgG concentration (mg/dL) using a standard curve and corrected for dilution factor of 400 000. To validate IgG ELISA, total IgG levels were compared for 8 North American pregnant women (Hawaii Biospecimen Repository) and 6 Cameroonian women [32 (link)] in the ELISA assay and by nephelometry (Clinical Labs of Hawaii).
8
Cytokine Profiles in COVID-19 Hospitalization
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To determine cytokine profiles of alive and death cases due to COVID-19 up to ten days of hospitalization, the plasma samples were isolated from whole blood of patients (at the first day of hospitalization) and healthy subjects. The levels of IL-1α, IL-1β, TNF-α, IL-6, TGF-β1, and IL-10 cytokines were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Mabtech, Sweden) according the manufacturer’s instructions. Turbidimetric immunoassay CRP of COVID-19 patients was performed by the Mindray BS-800 automated biochemistry analyzer (Shenzhen Mindray Bio-Medical Electronics, China). The level of erythrocyte sediment rate (ESR) of blood samples was measured using the erythrocyte sedimentation rate (ESR) analyzer (Parsian Teb, Iran).
9
Cytokine-Mediated T-Cell Activation Assay
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Peripheral blood was diluted at a ratio of 1:1·5 with RPMI 1640 Glutamax medium (ThermoFisher Scientific, Carlsbad, CA) and supplemented with antibiotics (penicillin, 100 IU/mL and streptomycin, 100 μg/mL) (ThermoFisher Scientific, Carlsbad, CA). Next, the diluted blood was treated either with i) human IL-7 (10 ng/mL) and IL-2 (500 IU/mL), or ii) human IL-2 (1000 IU/mL), IL-15 (10 ng/mL) and IL-21 (10 ng/mL) or left untreated (RPMI medium only). All cytokines were purchased from Prospec (Ness-Ziona, Israel). The cytokine-treated as well as the untreated blood samples were added to 96-well round-bottom plates pre-coated with a panel of antigens (Supplementary Table 1), followed by incubation at 37 °C with 5% CO2 for seven days. Antigen-free medium was used as negative control while 5 μg/mL phytohaemagglutinin (PHA, Sigma-Aldrich, St. Louis, MO), 30 ng/mL OKT3 (anti-human CD3 monoclonal antibody, BioLegend, CA) or 10 ng/mL SEA + SEB (Staphylococcal Enterotoxin A and B, Sigma-Aldrich, St. Louis, MO), respectively were used as positive controls. Measurement of IFN-γ production by immune cells after antigen exposure was performed with a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (MABTECH, Stockholm, Sweden).
10
Postprandial Lipoprotein Metabolism Analysis
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The quantification of analytes including lipids, lipoproteins, and apolipoproteins were detailed previously.29 (link) VLDL-apoB-100 from the TRL fraction was measured using an ELISA kit (Mabtech, Nacka, Sweden). Plasma apoB-48 levels were measured with an enzyme immunoassay kit (Shibayagi, Shibukawa, Japan). Fasting plasma HL (hepatic lipase; MyBioSource, San Diego, CA), LPL (Cusabio, Wuhan, China), and ANGPTL3 (R&D Systems) levels were determined using enzyme immunoassay kits. Postprandial metabolism was quantified by calculating the incremental AUC for plasma triglycerides, VLDL-apoB-100, and apoB-48 (0–10 h), using the trapezium rule. The incremental AUC was estimated as the difference between the area defined below the baseline concentration and the area under the plasma curve between hours 0 and 10. These measures provide an integrated estimate of the total dynamic response of TRL particles to a fat load.33 (link) Total AUC reflects exposure and potential impact of accumulation of TRLs on the artery and hence atherosclerotic CVD. By contrast, the incremental AUC reflects the acute change in TRLs after a fat load. Total AUC under TRL response curve has been shown to be a better predictor than fasting concentration or incremental AUC in predicting cardiovascular events.34 (link)
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