Plasma TG, FFA, total cholesterol (CHOL), high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were individually measured using enzymatic reagents (Diasys Diagnostic Systems, Holzheim, Germany). Glucose was measured using the oxidase method (Yellow Springs, OH, USA). Porcine ELISA kits were used to assay plasma levels of IL-6 and TNF-α (R&D Systems, Minneapolis, MN, USA). All samples were measured in triplicate in a single assay according to the manufacturers’ instructions.
Porcine elisa kit
Manufactured by R&D Systems
Sourced in United States
Porcine ELISA kits from R&D Systems are quantitative sandwich enzyme-linked immunosorbent assays designed for the measurement of porcine proteins in cell culture supernates, serum, and plasma samples.
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Lab products found in correlation
10 protocols using porcine elisa kit
1
Plasma Biomarkers Measurement Protocol
2
Porcine Serum Cytokine Profiling
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Blood samples were collected from 2 pigs per pen on d 6. Blood was collected from the cranial vena cava via a 20 gauge needle and a 10 mL serum vacutainer tube (BD, with clot activator and gel for serum separation). Tubes were inverted and allowed a minimum of 30 min to clot. Samples were centrifuged at 1,600×g for 10 min at 2 °C, and the separated serum samples were stored at −80 °C until analysis was performed. Serum concentrations of interleukin 6 (IL-6) and interleukin 8 (IL-8) were determined via porcine ELISA kits (R&D Systems, Minneapolis, MN). The minimum detection for IL-6 was 18.8 pg/ml and 62.5 pg/ml for IL-8. Assays were conducted as outlined by the manufacturer.
3
Measuring Porcine TNF-α in PBMCs
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The concentration of TNF-ɑ in the supernatants of PBMCs was measured using commercially available porcine ELISA kits (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instruction.
4
Porcine Cytokine Profiling with ELISA and Luminex
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Porcine ELISA kits from R&D (Minneapolis, MN), and two luminex-based assays: porcine 13-plex for Millipore (Billerica, MA), and porcine 9-plex from Invitrogen (Thermo Fisher Scientific Inc., Waltham, MA) were used. All analyses were carried out according to the manufacturer's kit instructions. Samples out of reading range were set to the upper detection limit, UDL. Blood samples were processed to either EDTA-plasma or serum. The IL-1β and IL-8 ELISA were carried out using serum, all other with EDTA-plasma, as recommended.
5
Cytokine Profiling in Porcine Study
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Concentrations of TNF-α, IL-1β, and IL-10 were measured in culture supernatants using porcine ELISA kits (R&D Systems, Lille, France). For each measure a ROUT test was performed to exclude animals from the analysis, if relevant.
6
ELISA Quantification of TNF-α and IL-1β
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The concentrations of TNF-α and IL-1β in supernatants of PBMCs were measured
using commercially available porcine ELISA kits (R&D Systems, Inc.,
Minneapolis, MN) according to the manufacturer’s instructions.
using commercially available porcine ELISA kits (R&D Systems, Inc.,
Minneapolis, MN) according to the manufacturer’s instructions.
7
Measuring TNF-α in Porcine Plasma
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The TNF-α concentration in plasma was measured by using a commercially available porcine ELISA kit (R&D Systems, Minneapolis, MN, USA, REF: PTA00)40 (link).
8
Biochemical Markers in Umbilical Vein Serum
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The malondialdehyde (MDA) concentration in the skeletal muscle was measured using an assay kit (Jiancheng Institute of Bioengineering, Nanjing, Jiangsu, China). The concentrations of triglyceride and glucose in umbilical vein serum were determined using commercial kits (Jiancheng Institute of Bioengineering, Nanjing, Jiangsu, China) according to the provided instructions. Growth hormone (GH) concentration in umbilical vein serum was measured using a commercially available radioimmunoassay kits purchased from Beijing North Institute of Biotechnology (Beijing, China). Insulin was determined with porcine ELISA kit (R&D Systems, Minneapolis, MN, USA). Sensitivities of the assays were 0.02 ng/ml and 2.15 pmol/l for GH and insulin, respectively. Intra- and inter-assay coefficients of variation were 4.3 and 6.6 % for GH and 3.9 and 7.4 % for insulin, respectively.
9
Swine Immune Responses Post-Weaning
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On day 0, 7, and 14 after weaning, whole blood samples of one randomly selected pig in each pen were collected by vacuum tubes with EDTA (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA), and the number of white blood cells (WBC) were analyzed by a hematology analyzer (scil Vet abc hematology analyzer; scil animal care company, F-67120 Altorf, France). Using serum samples on day 0, 7, and 14 after weaning, the porcine ELISA kit (R&D Systems Inc., Minneapolis, MN, USA) was used to measure the immune responses such as cortisol, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β), and interleukin-6 (IL-6). All cytokine measurements from serum samples were obtained according to a previously reported method [17 (link)]. The intra-assay coefficients of variation for cortisol, TNF-α, TGF-β1, IL-1 β, and IL-6 were 9.2, 6.9, 2.9, 7.2, and 5.1%, respectively, while the respective inter-assay coefficients of variation were 21.2, 9.2, 9.1, 8.7, and 7.4%.
10
Porcine Intestinal TNF-alpha ELISA
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Intestinal mucosa TNF-a concentration was analyzed using a commercially available porcine ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Briefly, the 100 ml of standard (23.4, 46.9, 93.8, 188, 375, 750, 1500 pg/ml TNF-a), control or diluted intestinal samples were added to a 96-well microtiter plate and incubated at room temperature (21-25 C) for 2 h. Following incubation, each well was aspirated and the plate was washed five times. After washing, a 100 -ml solution of conjugate was added to the wells. The plate was incubated at room temperature for 2 h and then washed five times. After that, 100 ml substrate solution was added. After incubation for 30 min at room temperature, 100 ml stop solution was added, and the plates were read at 450 nm using an ELISA plate reader (Model 550; Bio-Rad, Hercules, CA, USA). Results obtained using natural porcine TNF-a show dose-response curves that are parallel to the standard curves. The minimum detectable concentration was 3.7 pg/ml. The TNF-a concentration in intestine was expressed as pg/mg protein.
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