To detect anti-avian HEV IgG antibodies in serum samples collected each week, an indirect ELISA using a truncated recombinant avian HEV ORF2 protein as the coating antigen was performed as previously described (Zhao et al., 2013 (link)). Briefly, 96-well plates were coated with the purified target protein. After the wells were blocked and washed, 1:50 serial dilutions of serum samples (100 μL/well) were added, and the plates were incubated for 1 h at room temperature (RT ). Next, horseradish peroxidaseconjugated goat anti-chicken IgG diluted 1:4000 (100 μL/well) was added. After 3 washes, tetramethylbenzidine (TMB ) was added to each well and the plates were incubated in the dark for 15 min at RT. The colorimetric reaction was stopped by adding 3 M H2SO4 (50 μL/well) and the optical density (OD) values were read at 450 nm (OD450nm) using an automated ELISA plate reader (Bio-Rad, Hercules, CA). Based on the indirect ELISA assay, the result was considered positive when the OD450nm of testing serum samples was greater than 0.368.
Automated elisa plate reader
Manufactured by Bio-Rad
Sourced in United States
The Automated ELISA Plate Reader is a laboratory instrument used to measure the absorbance of samples in multi-well microplates. It is designed to perform enzyme-linked immunosorbent assay (ELISA) tests automatically, providing accurate and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using automated elisa plate reader
1
Indirect ELISA to Detect Avian HEV IgG
2
Production and Detection of Nanobody-HRP Fusions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The platform for the production of nanobody-HRP fusion proteins was constructed as previously described [26 (link)]. PCV2-Cap protein nanobody (Nb15) gene fragment was inserted between the HA tag and HRP sequences of the novel vector pCMV-N1-HRP. The positive recombinant plasmids were confirmed by sequencing and named as RANbodies-PCV2-Nb15-HRP. To produce nanobody-HRP fusion protein, the mammalian cell line HEK 293T cells were transfected with RANbodies-HRP-PCV2-Nb15 plasmid using polyetherimide (PEI, Polysciences Inc. Warrington, USA) reagent. After the cells were transfected for 3 days, the medium containing secreted nanobody-HRP fusion protein was harvested and filtered through 0.45 µm pore cellulose acetate membranes for direct use (Scheme 1a ).
To detect the titer of the nanobody-HRP fusion protein in the medium, the filtered medium was directly used for iELISA. Briefly, the wells of the ELISA plate were coated with 400 ng/well purified PCV2-Cap protein overnight at 4 °C, after blocking and washing, a diluted medium was added and incubated. After washing, tetramethylbenzidine (TMB) was added for a colorimetric reaction at RT for 15 min. Finally, the colorimetric reaction was stopped by adding 3 mol/L H2SO4 (50 µL/well), and the OD450nm values were read using an automated ELISA plate reader (Bio-Rad, USA).
To detect the titer of the nanobody-HRP fusion protein in the medium, the filtered medium was directly used for iELISA. Briefly, the wells of the ELISA plate were coated with 400 ng/well purified PCV2-Cap protein overnight at 4 °C, after blocking and washing, a diluted medium was added and incubated. After washing, tetramethylbenzidine (TMB) was added for a colorimetric reaction at RT for 15 min. Finally, the colorimetric reaction was stopped by adding 3 mol/L H2SO4 (50 µL/well), and the OD450nm values were read using an automated ELISA plate reader (Bio-Rad, USA).
3
Anti-avian HEV IgG Antibody Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-avian HEV IgG antibodies were detected in both clinical and experimental serum samples using an indirect ELISA described previously by Zhao et al. [16 (link)]. Briefly, a purified truncated recombinant CaHEV capsid protein expressed in Escherichia coli was used as the coating antigen for the indirect ELISA. After the coated plates were blocked and washed, the serum samples (100 μL/well) were added into the wells and incubated for 1 h at room temperature (RT). After three washes, a horseradish peroxidase-goat anti-chicken IgG diluted 1:4000 (100 μL/well) was added to the wells and incubated for 1 h again at room temperature. After a final three washes, 3,3′,5,5′-tetramethylbenzidine (TMB) was added to each well and the plates were incubated in the dark for 15 min at RT. The colorimetric reaction was stopped by adding 3 M H2SO4 (50 μL/well) and the optical density (OD) values were read at 450 nm using an automated ELISA plate reader (Bio-Rad, USA). All sera were tested in at least duplicate wells.
4
Horseradish Peroxidase Labeling of mAbs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mAbs were labeled with horseradish peroxidase (HRP, Sigma, Missouri, USA) by the sodium periodate oxidation method described by Kanpp [19] (link). The titers of the labeled mAbs were determined by indirect ELISA. The 96-well microtiter plates (Maxisorp Nunc, Denmark) were coated with purified capture mAbs diluted in sodium carbonate buffer. TMUV was added as antigen and the plates were incubated for 30 min at 37°C. Then mAbs conjugated with horseradish peroxidase (detection antibody) were added. The unbound conjugates were washed off after incubation, and 3, 3', 5, 5'-Tetramethylbenzidine (TMB) substrate solution (TIANGEN, Beijing, China) was added to each well. Incubation was carried out for 30 min and the reaction was stopped by adding 3M H2SO4. Plates were read at 450 nm on an automated ELISA plate reader (Bio-Rad, USA). The best pairing antibodies were obtained according to the recorded result.
5
Serological Testing for Avian Viruses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine whether chickens had been exposed to ALV-J, all serum samples were tested for anti-ALV-J antibodies using a commercial ELISA test kit (FlockChek@ Avian Leucosis Virus Antibody Test Kit-Subgroup J, IDEXX, FlockChek@) according to the manufacturer’s instructions. Absorbance values were measured using an automated ELISA plate reader (Bio-Rad, USA) and the results were analyzed and reported as positive or negative based on the cut-off value listed in the manufacturer’s instructions.
Anti-avian HEV IgG antibodies in the serum samples were tested using iELISA developed by Qin et al. [20 (link)]. It employs a recombinant truncated avian HEV ORF2 protein containing the C-terminal 268 amino acids as the coating antigen. The recombinant avian HEV ORF2 protein was expressed in E. coli and purified using the BugBuster Ni-NTA His•Bind Purification Kit (Jinsite Co., China). The ORF2 gene originates from a Chinese isolate of avian HEV (CaHEV genotype 3, GenBank number: GU954430). All serum samples were tested in duplicate for each of the two iELISAs to test for anti-ALV-J and anti-avian HEV antibodies.
Anti-avian HEV IgG antibodies in the serum samples were tested using iELISA developed by Qin et al. [20 (link)]. It employs a recombinant truncated avian HEV ORF2 protein containing the C-terminal 268 amino acids as the coating antigen. The recombinant avian HEV ORF2 protein was expressed in E. coli and purified using the BugBuster Ni-NTA His•Bind Purification Kit (Jinsite Co., China). The ORF2 gene originates from a Chinese isolate of avian HEV (CaHEV genotype 3, GenBank number: GU954430). All serum samples were tested in duplicate for each of the two iELISAs to test for anti-ALV-J and anti-avian HEV antibodies.
6
Production and Characterization of PCV2-Nanobody-HRP Fusion Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The platform for production nanobody-HRP fusion protein was constructed as previously described [26] . PCV2-Cap protein nanobody (Nb15) gene fragment was inserted between the HA tag and HRP sequences of the novelty vector pCMV-N1-HRP. The positive recombinant plasmids were con rmed by sequencing and named as RANbodies-PCV2-Nb15-HRP. To produce nanobody-HRP fusion protein, the mammalian cell line HEK 293T cells were transfected with RANbodies-HRP-PCV2-Nb15 plasmid using polyetherimide (PEI, Polysciences Inc. Warrington, USA) reagent. After the cells were transfected for 3 days, the medium containing secreted nanobody-HRP fusion proteins was harvested and ltered through 0.45 μm pore cellulose acetate membranes for direct use (Scheme 1a).
To detect the titer of the nanobody-HRP fusion protein in the medium, ltered medium was directly used for iELISA. Brie y, the wells of the ELISA plate were coated with 400 ng puri ed PCV2-Cap protein overnight at 4 °C, after blocking and washing, a diluted medium was added and incubated. After washing, tetramethylbenzidine (TMB) was added for a colorimetric reaction at RT for 15 min. Finally, the colorimetric reaction was stopped by adding 3 mol/L H 2 SO 4 (50 µL/well), and the OD 450nm values were read using an automated ELISA plate reader (Bio-Rad, USA).
To detect the titer of the nanobody-HRP fusion protein in the medium, ltered medium was directly used for iELISA. Brie y, the wells of the ELISA plate were coated with 400 ng puri ed PCV2-Cap protein overnight at 4 °C, after blocking and washing, a diluted medium was added and incubated. After washing, tetramethylbenzidine (TMB) was added for a colorimetric reaction at RT for 15 min. Finally, the colorimetric reaction was stopped by adding 3 mol/L H 2 SO 4 (50 µL/well), and the OD 450nm values were read using an automated ELISA plate reader (Bio-Rad, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!