iELISA was performed to measure PCV-2-specific IgG levels in serum and IgA concentrations in saliva as well as lung and intestinal lavage (mucosal) samples [29 ]. In brief, 96-well ELISA plates (Corning, USA) were coated with 0.2 µg PCV-2 antigen in 0.05 M carbonate buffer (pH 9.6) per well and incubated overnight at 4℃. After blocking with 5% skimmed milk (Bio-Rad, USA) in PBS plus 0.5% Tween 20 (PBST) at 37℃ for 1 h, 100 µL of serum sample diluted 200-fold or undiluted mucosal samples were added to each well and the plates were incubated at 37℃ for 1 h. The plates were then washed three times with PBST to remove unbound antibodies. Horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (1 : 2,000; Baosen Biotechnology, China) or HRP-conjugated goat anti-mouse IgA (1 : 10,000; Bethyl Laboratories, USA) was subsequently added to each well. After incubating at 37℃ for 1 h, the plates were washed three times and developed with 3,3',5,5'-tetramethylbenzidine (TMB; Sigma, USA) for 15 min. The reaction was stopped by adding 1 M H2SO4, and the optical density (OD) was measured at a wavelength of 450 nm using a Thermo plate reader (Thermo Scientific, USA).
Thermo plate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo plate reader is a versatile instrument designed for a variety of absorbance-based assays in microplates. It measures the optical density of samples in microplate wells, which can be used to quantify the concentration or activity of various analytes.
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2 protocols using thermo plate reader
1
PCV-2-Specific Antibody Detection by iELISA
2
Cholinesterase Activity Assay in Drosophila
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The AChE and BChE activities were measured as described by Ellman et al. 21 and modified by Silva de Sá et al. 22 For that, 100 flies (Drosophila melanogaster) were anaesthetized in ice and solubilized in 1 mL of tris-HCl solution (0.05 M, pH 7.4); the mixture was centrifuged at 12 000 rpm for 10 min, and then the supernatant was collected and stored in an ultrafreezer (-80 °C) for future analysis of cholinesterase activity. Analysis was performed in duplicate using a reaction medium that contained 100 µL of potassium phosphate buffer (TFK, 100 mM, pH 7.5), 20 µl of water, 10 µL of supernatant, and 20 µL of extract (at final concentrations of 20, 14, 8, and 2 mg mL -1 ); a control was also studied (without the addition of extract). First, the medium was incubated at 30 °C for 5 min, and then 20 µL of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 20 µL of acetylthiocholine were added in the dark (for BChE tests, this volume was replaced with the same volume of butyryl thiocholine). Absorbance determinations were performed every minute (for 4 min) in a plate reader (Thermo-Plate Reader) at a wavelength of 405 nm. The enzymatic activity was expressed as a percentage of activity relative to the control group (100%).
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