Surrogate virus neutralization assay, which allows assessment of virus inhibition without the BSL-3 facility requirement, was performed as described previously [25 (link)]. HRP-conjugated RBD was provided by Optipharm (Cheongju, Republic of Korea), which was performed using the HRP conjugation kit (Abcam, Cambridge, UK). SARS-CoV-2 human angiotensin-converting enzyme 2 (hACE2) receptor protein was purchased from GenScript (Piscataway, NJ, USA). After coating the plates with 100 ng of hACE2 using carbonate coating buffer in 96-well plates overnight at 4 °C, plates were washed with PBST and subsequently blocked with BD OptEIA assay diluent (BD Bioscience, San Diego, CA, USA) for 1 h at RT. Sera collected from mice 1 week after the final immunization were serially diluted in PBS and incubated at 56 °C for 30 min for complement inactivation. After heat inactivation, equal volumes of inactivated sera and HRP-conjugated RBD were mixed and inoculated into each well to be incubated for 1 h at 37 °C. Wells were washed with PBST and chromogenic reactions were developed using TMB substrate (BD Bioscience, San Diego, CA, USA). Reactions were stopped with 2N H2SO4 and absorbance values at 450 nm were measured. Percentage of inhibition was calculated as follows: Inhibition (%) = (1 − sample OD450/control OD450) × 100.
Opteia assay diluent
Manufactured by BD
Sourced in United States, China
BD OptEIA assay diluent is a laboratory reagent used to dilute samples in enzyme-linked immunosorbent assay (ELISA) tests. It is designed to maintain the optimal conditions for antibody-antigen interactions during the ELISA procedure.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by Bio-Rad (2 mentions)
Wellnunc maxisorp flat bottom plates, by Thermo Fisher Scientific (2 mentions)
Biotin sp conjugated goat anti mouse ige, by Southern Biotech (2 mentions)
Imark microplate reader, by Bio-Rad (2 mentions)
Streptavidin hrp, by BD (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (2 mentions)
Maxisorp plate, by Thermo Fisher Scientific (2 mentions)
Hace2 protein, by Sino Biological (2 mentions)
Hrp conjugation kit, by Abcam (1 mentions)
Tmb substrate, by BD (1 mentions)
Sars cov 2 rbd protein, by GenScript (1 mentions)
Phosphatase substrate, by Merck Group (1 mentions)
Zonulin, by MyBioSource (1 mentions)
Il 17a, by Thermo Fisher Scientific (1 mentions)
Softmax pro 6, by Molecular Devices (1 mentions)
Akp streptavidin, by BD (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Greiner (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Horseradish peroxidase conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
11 protocols using opteia assay diluent
1
Surrogate SARS-CoV-2 Neutralization Assay
2
Serum HDM-specific IgE ELISA Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum HDM-specific IgE ELISA was performed as described previously(15 (link)). 96-well
Nunc MaxiSorp flat-bottom plates (Thermo-Fisher Scientific, Waltham, MA) were coated with 10 ug/mL HDM in sodium bicarbonate
buffer (pH 9.5) for 48 hours at 4°C. Following coating, plates came to room temperature for 15 minutes and were washed six
times with 0.05% PBS/Tween 20 (PBST) and blocked for one hour at 37°C with BD OptEIA Assay Diluent (BD
Biosciences, San Diego, CA; Cat #555213). After washes, serum samples were added in two-fold serial dilutions (range 1:20
to 1:2560) and incubated at room temperature for 1.5 hours.
Plates were washed eight times with PBST and labeled with Biotin-SP-conjugated goat anti- mouse IgE (Southern Biotech,
Birmingham, AL; Cat #1110-08) diluted 1:5000 in blocking buffer for one hour at room temperature. Plates were then washed
six times and incubated with streptavidin HRP (BD Biosciences, San Jose, CA; Cat #554066) diluted 1:1000 in blocking
buffer at room temperature for thirty minutes. Plates were washed seven times with PBST and incubated with
3,3',5,5'-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD; Cat #50-76-01) for 20 minutes in the dark at room temperature.
The reaction was stopped using 1 M phosphoric acid at equal volume to TMB and the plates were read at dual 450nm-570nm wavelengths
using an iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA).
Nunc MaxiSorp flat-bottom plates (Thermo-Fisher Scientific, Waltham, MA) were coated with 10 ug/mL HDM in sodium bicarbonate
buffer (pH 9.5) for 48 hours at 4°C. Following coating, plates came to room temperature for 15 minutes and were washed six
times with 0.05% PBS/Tween 20 (PBST) and blocked for one hour at 37°C with BD OptEIA Assay Diluent (BD
Biosciences, San Diego, CA; Cat #555213). After washes, serum samples were added in two-fold serial dilutions (range 1:20
to 1:2560) and incubated at room temperature for 1.5 hours.
Plates were washed eight times with PBST and labeled with Biotin-SP-conjugated goat anti- mouse IgE (Southern Biotech,
Birmingham, AL; Cat #1110-08) diluted 1:5000 in blocking buffer for one hour at room temperature. Plates were then washed
six times and incubated with streptavidin HRP (BD Biosciences, San Jose, CA; Cat #554066) diluted 1:1000 in blocking
buffer at room temperature for thirty minutes. Plates were washed seven times with PBST and incubated with
3,3',5,5'-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD; Cat #50-76-01) for 20 minutes in the dark at room temperature.
The reaction was stopped using 1 M phosphoric acid at equal volume to TMB and the plates were read at dual 450nm-570nm wavelengths
using an iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA).
3
SARS-CoV-2 RBD Protein ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
96-well Maxisorp plates (Nunc) were coated with 2 µg/ml of SARS-CoV-2 RBD protein (Genscript) in bicarbonate buffer overnight at 4°C. Wells were blocked using BD OptEIA assay diluent (BD) for 1 h at 37°C. Heat-inactivated sera were depleted for serum IgG using Gullsorb™ Human IgG Inactivation Reagent (Meridian Bioscience) as per manufacturer’s recommendations. Depleted and undepleted sera were then further diluted to a final concentration of 1:200, added into ELISA microwells and incubated for 1 h at 37°C. Following extensive washing, anti-human IgM-HRP (Life Technologies) or anti-human IgG-HRP (Santa Cruz) diluted 1:10,000 was added and incubated for 30 min at 37°C. The chromogenic reaction was quantified following the addition of TMB substrate (Invitrogen) and stop solution (KPL SeraCare). The absorbance of the samples was measured at 450 nm and the background at 570 nm. The results are presented as the OD difference of 450 and 570 nm.
4
Quantifying Cytokine and Autoantibody Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure IFN-γ and IL-17A levels in cell culture supernatants, 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with IFN-γ (eBioscience, San Diego, CA, USA) or IL-17A monoclonal antibodies (eBioscience) and blocked with BD OptEIA™ Assay diluent (BD Biosciences). Samples were added to the wells and incubated overnight at 4°C. Biotinylated IFN-γ (eBioscience) or IL-17A (eBioscience) antibodies were added, followed by the addition of AKP streptavidin (BD Biosciences). Color was developed by adding 5 mM phosphatase substrate (Sigma-Aldrich), and absorbance was measured at 405 nm using a Versamax microplate reader with SoftMax Pro 6.5.1 software (Molecular Devices, Wokingham, UK).
Serum samples were prepared by centrifuging whole blood collected from mice at 2,000 × g for 10 min at 4°C. Subsequently, the serum samples were subjected to dilutions of 100,000-fold, 4-fold, and 1000-fold for the detection of anti-CII IgG antibody (Chondrex), anti-CCP antibody (MyBioSource Inc., San Diego, CA, USA), and zonulin (MyBioSource Inc.), respectively. The levels of serum autoantibodies and zonulin (MyBioSource Inc.) were measured in accordance with the manufacturer’s instructions.
Serum samples were prepared by centrifuging whole blood collected from mice at 2,000 × g for 10 min at 4°C. Subsequently, the serum samples were subjected to dilutions of 100,000-fold, 4-fold, and 1000-fold for the detection of anti-CII IgG antibody (Chondrex), anti-CCP antibody (MyBioSource Inc., San Diego, CA, USA), and zonulin (MyBioSource Inc.), respectively. The levels of serum autoantibodies and zonulin (MyBioSource Inc.) were measured in accordance with the manufacturer’s instructions.
5
Serum HDM-specific IgE ELISA Protocol
6
SARS-CoV-2 IgM/IgG Antibody ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
96-well Maxisorp plates (Nunc) were coated with 10 µg/ml of anti-human IgM (SeraCare) or anti-human IgG (Jackson labs) in bicarbonate buffer overnight at 4°C. Wells were blocked using BD OptEIA assay diluent (BD) for 1 h at 37°C and heat-inactivated sera diluted 1:50 were next added and incubated for 1 h at 37°C. Following extensive washing, SARS-CoV-2-HRP (GenScript) diluted 4 µg/ml was added and incubated for 30 min at 37°C. Chromogenic reaction was quantified following the addition of TMB substrate (Invitrogen) and stop solution (KPL SeraCare). The absorbance of the samples was measured at 450 nm and the background at 570 nm. Negative controls consisting of 37 naïve human sera were added in the assay. The results are presented as fold change over average reading of negative controls.
7
Quantifying Serum IgG Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure total IgG levels in the serum, plates were coated with 2 µg/ml of donkey anti-mouse IgG (Affinipure; Jackson ImmunoResearch Laboratories, West Grove, PA)–PBS, washed in PBS with 0.05% Tween 20, and blocked in 1% bovine serum albumin (BSA)–PBS prior to incubation with serial dilutions of serum or the mouse IgG standard (ChromPure; Jackson ImmunoResearch) in assay diluent (OptEIA assay diluent; BD Biosciences) overnight at 4°C. IgG was detected by the use of horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch), R & D Systems (Minneapolis, MN) substrate, and stop solution, and absorbance at 450 nm was read on a FilterMax5 microplate reader (Molecular Devices, Sunnyvale, CA). To measure levels of IgG specific for viral antigens, plates were first coated with 0.5% paraformaldehyde-fixed viral antigen–PBS overnight at 4°C.
8
Indirect ELISA for Detecting Anti-H1N1 Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant influenza A H1N1 (A/Puerto Rico/8/1934) hemagglutinin and influenza A H1N1 (A/Puerto Rico/8/1934) hemagglutinin-specific mouse monoclonal antibody were purchased from Sino Biological (Beijing, China).
For indirect enzyme-linked immunosorbent assay (ELISA), 1 µg of recombinant hemagglutinin protein was coated onto a 96-well microplate in 100 mM carbonate-bicarbonate buffer (pH 9.6) and blocked with OptEIA Assay Diluent (55,213, BD Biosciences, Franklin Lakes, NJ, USA) for 1 h at room temperature. Following five washes with 1× PBS, plasma from PR8 virus-infected mice was added and incubated for 2 h at room temperature. Unbound antibodies were eliminated by washing five times with 1× PBS containing Tween 20. The signal was developed based on the enzymatic reaction of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (555,214, BD Biosciences). The absorbance at 450 nm was assessed and the background absorbance at 570 nm was measured.
For indirect enzyme-linked immunosorbent assay (ELISA), 1 µg of recombinant hemagglutinin protein was coated onto a 96-well microplate in 100 mM carbonate-bicarbonate buffer (pH 9.6) and blocked with OptEIA Assay Diluent (55,213, BD Biosciences, Franklin Lakes, NJ, USA) for 1 h at room temperature. Following five washes with 1× PBS, plasma from PR8 virus-infected mice was added and incubated for 2 h at room temperature. Unbound antibodies were eliminated by washing five times with 1× PBS containing Tween 20. The signal was developed based on the enzymatic reaction of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (555,214, BD Biosciences). The absorbance at 450 nm was assessed and the background absorbance at 570 nm was measured.
9
SARS-CoV-2 Neutralizing Antibody Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The surrogate virus based neutralization test (sVNT) was used for measuring neutralizing antibody (sNAb) to SARS-CoV-2 as previously described [25 (link)]. Briefly, microtiter plates were coated overnight with 2 μg/ml hACE2 protein (Sino Biological, China) at 4°C, followed by blocking with OptEIA assay diluent (BD). The HRP–RBD conjugate (3 ng) was pre-incubated with 100 μl of diluted serum samples for 1 h at 37°C, then added into hACE2 pre-coated plate for 1 h at room temperature. Plates were washed five times by PBST. A colorimetric signal was developed with TMB, and equal volume of stop solution was added to terminate the reaction. The absorbance reading was performed at 450 nm and 570 nm. Inhibition rate (%) = (1 − sample optical density value/negative control optical density value) × 100.
10
Bat IL-1β Sandwich ELISA Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ELISAs for human/mouse IL-1β in cell-free supernatants were performed according to the manufacturer’s instructions (R&D Systems; DY201 and DY401). Sandwich ELISA for bat IL-1β was generated using the goat anti-dog IL-1β (ab193852) as the capturing antibody and rabbit anti-mouse IL-1β (ab9722) as the detection antibody. Bicarbonate/carbonate coating buffer (50 mM), OptEIA assay diluent (BD bioscience), donkey anti-rabbit HRP-conjugated secondary antibody (Santa Cruz), 3,3′,5,5′-tetramethylbenzidine (TMB) chromogen solution (Invitrogen) and TMB stop solution (VWR) were used in the assay. Purified P. alecto IL-1β-Fc recombinant proteins were used as standards.
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!