Macromolecules [thyroglobulin (Tg), TIC, TIM, guar galactomannan or yeast glycoproteins] were coated on Nunc MAXISORB microplate wells (maximum 1 µg in 200 µl PBS) by incubation at 37˚C for 3 h or overnight at 4˚C. The wells were washed with PBS containing 0.05% Tween-20 (PBST) and blocked by incubation for 30 min at 37˚C with PBS containing 0.5% Tween-20. After another wash with PBST, the wells were incubated for 2 h at 4˚C with 200 µl PBST containing the primary reactants (sugar-extracted platelet-bound triplets or antibodies separated from them) pre-incubated with or without specific sugars. Wells were washed with PBST again and treated with HRP-labelled secondary reactants (anti-immunoglobulin antibodies for antibody assay or anti-albumin antibody for triplet assay). After washing the plates again three times with PBST, bound HRP was measured by adding 200 µl OPD (0.5 mg/ml) dissolved in 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.03% H2O2 for 15 min at 25˚C, followed by the addition of 50 µl 12.5% H2SO4 to stop the reaction. Absorbance was measured at 490 nm using an ELx800™ ELISA reader (BioTek Instruments, Inc.).
Elx800 elisa reader
Manufactured by Agilent Technologies
Sourced in United States
The ELX800 ELISA reader is a compact and versatile microplate reader designed for absorbance-based ELISA (Enzyme-Linked Immunosorbent Assay) measurements. It is capable of reading 96-well microplates and supports a range of wavelengths for various ELISA applications.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
96 well plate, by Corning (2 mentions)
Maxisorb, by Thermo Fisher Scientific (1 mentions)
Id screen paratuberculosis indirect, by IDvet (1 mentions)
Rhuepo, by R&D Systems (1 mentions)
Soluble epor, by R&D Systems (1 mentions)
Reverse transcriptase assay kit, by Roche (1 mentions)
Ortho phenylenediamine, by Merck Group (1 mentions)
Vacutainer sst tube, by BD (1 mentions)
Bio tek elx50, by Agilent Technologies (1 mentions)
Advia 2400, by Siemens (1 mentions)
Rat elisa kit, by Abcam (1 mentions)
Mem g9 mab, by Exbio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Anti pi3k, by ABclonal (1 mentions)
Anti mtor, by ABclonal (1 mentions)
37 protocols using elx800 elisa reader
1
Glycoprotein-Mediated Antibody Binding Assay
2
Quantifying Anti-HBsAg Antibodies by ELISA
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The immunopotentiation was measured by quantifying specific antibodies with a rat anti-HBsAg IgG enzyme-linked immunosorbent test kit (catalogue number 4270) from Alpha diagnostic International, United States. The test is based on the binding of rat anti-HBsAg in the serum samples to HBsAg immobilized on microwells of a microtiter plate. By the addition of horse radish peroxidase enzyme, the substrate tetramethyl benzidine (TMB), and stopping solution. The intensity of the color developed was measured by determining its absorbance at 450 nm using a BioTek ELx 800 ELISA reader (BioTek, Winooski, VT, USA). The concentration of anti-HBsAg was determined by extrapolating on the standard curve.
3
Glutathione Quantification in Brain and Spinal Cord
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The glutathione contents of the brain and spinal cord were assessed using the Ellman reagent. Equal amounts of homogenized tissues and 10% w/v trichloroacetic acid were mixed. After centrifugation, 500 μl of supernatant was mixed with reaction buffer containing 3 ml of phosphate buffer (0.3 M, pH 8.9) and 500 μl of dithiobisnitrobenzoic acid (DTNB) (0.01 M). The absorbance of yellow colour was read at 412 nm using BioTek ELX800 ELISA Reader, USA [25 (link)].
4
Serological Detection of Anti-Map Antibodies
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Serological testing for production of antibodies (Ab) against Map was determined in all animals of the study using an indirect ELISA validated for domestic cattle, ID Screen® Paratuberculosis Indirect (IDVet, Grabels, France). This ELISA has a reported sensitivity of 41.5% and specificity of 99.42% [31 (link),32 ]. The test is a Mycobacterium phlei absorbed ELISA detecting anti-Map immunoglobulin G. The technique was performed according to the manufacturer’s instructions for bovine samples. The absorbance values were measured spectrophotometrically at 450 nm using an ELX800 ELISA reader (Bio-Tek Instruments, Winooski, VT, USA). The results were expressed as a quotient between the mean OD of each sample serum and the mean OD of the positive control serum in each plate.
5
Quantification of Interleukin-2 by Sandwich ELISA
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The sandwich ELISA was used to quantify interleukin 2 in tissue samples. The assay employs a monoclonal antibody specific to IL-2 coated on a 96-well assay plate. The standard and sample were analysed by binding with immobilized antibody. The end reaction was quantified by the intensity of colour formation which was measured at 450 nm by using BioTek ELX800 ELISA reader. The sample concentration was calculated by extrapolating on the standard curve.
6
Glucose and Insulin Quantification Protocol
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Glucose: Glucose was measured in serum by the method of glucose oxidase [16 (link)] using Crescent diagnostics kit, Jeddah, Saudi Arabia.
Insulin: The insulin was estimated by simple step sandwich ELISA technique. The free insulin is quantified by end point determination. The end point was estimated by the development of yellow colour which was measured at 450 nm by using Bio Tek EL X 800 ELISA reader. The sample concentration was calculated by extrapolating on the standard curve.
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7
Quantitative Caspase-9 Apoptosis Assay
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Caspase 9 is an apoptotic marker which was determined in tissue sample by spectrophotometric detection using chromophore p-nitroaniline (p-NA) after cleaving the labelled substrate LEHD-p-NA. The end reaction was quantified by detecting free p-NA after cleavage from the substrate, which was measured at 405 nm by using BioTek ELX800 ELISA reader. The sample concentration was calculated by extrapolating on the standard curve.
8
Quantitative IL-1β ELISA Assay
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Interleukin 1β in tissue sample was assessed by a simple step sandwich ELISA assay as per standard protocol given by the kits. The end reaction was quantified by the intensity of colour formation which was measured at 450 nm by using BioTek ELX800 ELISA reader. The sample concentration was calculated by extrapolating on the standard curve.
9
Cytokine Quantification by ELISA
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IL1-β, IL-2 and TNFα were assessed by using simple sandwich ELISA technique using Abcam's assay kit. The development of yellow colour end point was considered as cytokine concentration. The end point was estimated by the development of yellow colour which was measured at 450 nm by using Bio Tek EL X 800 ELISA reader. The sample concentration was calculated by extrapolating on the standard curve.
10
Cell Proliferation Assay via MTT
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Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT, Sigma-Aldrich). Cells were seeded in a 96-well microplate (4500 cells/well) and incubated at 37 °C overnight before rHuEPO (R&D Systems, Minneapolis, USA) or soluble-EPOR (R&D Systems, Minneapolis, USA) treatments. After culture finished, the cells were incubated with a medium containing 5 mg/mL MTT for 4 h at 37 °C. After precipitated formazan was dissolved in 150 ul DMSO, then the absorbance was detected on a BioTek Elx 800 ELISA reader (Winooski, VT, USA) at a wavelength of 570 nm.
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