As a reference method for the measurement of IFA, we used the commercial Anti-Intrinsic Factor ELISA (IgG) kit (EUROIMMUN, Lübeck, Germany), approved for diagnostic use. Following the manufacturer's instruction, we tested a subset of 45 serum samples from our cohort, comprising 33 patients with confirmed CAG and 12 subjects without CAG. Moreover, we tested the serial dilutions of two strongly IFA-positive samples in normal serum, of which one is used as standard curve in the LIPS assay. The absorbance of samples and the background were read at 450 nm and 655 nm, respectively, in an ELISA 680 Microplate reader (Bio-Rad). IFA levels were calculated by interpolation of the absorbance (after subtraction of the background) using a standard curve consisting of three calibrators provided by the kit (corresponding to 200, 20 and 2 relative units/ml (RU/ml)). The ELISA threshold for positivity was set at ≥20 RU/ml, according to the manufacturer's instructions.
Elisa microplate reader 680
Manufactured by Bio-Rad
Sourced in United States
The ELISA microplate reader (680) is a versatile laboratory instrument designed for quantitative and qualitative analysis of enzyme-linked immunosorbent assays (ELISA). It features absorbance measurement capabilities across a broad wavelength range to facilitate various ELISA applications.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ckx41 32 inverted microscope, by Olympus (1 mentions)
0.22 mm nitrocellulose membranes, by Merck Group (1 mentions)
Sp2 laser scanning confocal microscope, by Leica (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Epics xl flow cytometer, by Beckman Coulter (1 mentions)
Electrophoresis system, by Bio-Rad (1 mentions)
Whatman 1 filter paper, by Cytiva (1 mentions)
5 protocols using elisa microplate reader 680
1
ELISA-based IFA Measurement in CAG
2
Cell Culture and Imaging Protocols
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The following instruments and materials were used in the experimental procedures: CO-150-type CO 2 cell culture incubator (NBS Co., USA), EPICS XL flow cytometer (Beckman Coulter Co., USA), CKX-41-32 inverted microscope (Olympus, Japan), fluorescence microscope (Leica, Germany), SW-CJ-2f super clean workbench (Suzhou Purification Plant, China), ELISA 680 microplate reader (Bio-Rad, USA), SP-2 confocal laser scanning microscope (Leica), 0.22-mm nitrocellulose membranes (Sigma-Aldrich), electrophoresis system (Bio-Rad, 165-8001), GIS-2019 gel imaging system (Tanon Science & Technology Co., Ltd., China), automatic pipette (Gilson, France).
3
Mycotoxin Analysis in South Korean Feeds
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All samples were analyzed by Quantas Analytics, a subsidiary company of Romer Labs Diagnostic GmbH, which specializes in feed analysis. Afla, OTA, DON, ZEN, FUM, and T-2 extractions were performed according to the manufacturer’s instructions for enzyme-linked immunosorbent assays (ELISA). Twenty grams of sample were extracted with 100 ml of 70% methanol for Afla, ochratoxin, ZEN, and T-2 and with 100 ml of distilled water for DON. The extracts were filtered, and 1 ml of filtrate was diluted with 1 ml of distilled water except for DON, for which no dilution was made. The extract was filtered through a Whatman #1 filter paper, and the filtered sample was then diluted with distilled water. About 100 μl of diluted filtrate per well was used for the ELISA test. The optical density was measured at a wavelength of 450 nm using an ELISA microplate reader 680 (USA, Bio-Rad) with RIDASCREEN software for evaluation of ELISA data and the mycotoxin concentrations. To get a better overview of mycotoxin occurrence in South Korea, data were analyzed as follows: geographical regions, means of growth stages of pig production, and feeder types.
4
Cell Proliferation Assays: MTT and EdU
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Cell proliferation was measured by using MTT assay. After transfection, 3 × 103 per well were seeded in 96-well plates, and viability was assessed in six replicates at 24, 48 h, and 72 h. 20 μl MTT was added, and the mixture was incubated for 4 h at 37°C. Subsequently, 150 μl dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO, USA) was added to each well, followed by thorough mixing for 10 min. The absorbance was measured using an ELISA microplate reader (680; Bio-Rad, Hercules, CA, USA) at a wavelength of 490 nm. EdU incorporation assays were performed using Click-iT EdU Imaging Kit (US EVERBRIGHT INC.) according to the manufacturer's instructions.
5
MTT Assay for Cell Viability
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CNE2, 6-10B or 9-4E cells were seeded in 96-well plates (7×103 cells/well). Following transfection for various time periods (0, 24, 48 and 72 h), 20 µl MTT solution was added and the mixture was incubated for 4 h at 37°C. Subsequently 150 µl dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO, USA) was added to each well followed by thorough mixing for 10 min. The absorbance was measured using an ELISA microplate reader (680; Bio-Rad, Hercules, CA, USA) at a wavelength of 490 nm (with 630 nm as the reference wavelength).
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