Serum from all groups of mice were collected 3 weeks after booster vaccination and 5 days after ocular challenge infection. To determine the antigen specific antibody isotypes, 96-well plates (high protein binding plates; Corning, Inc) were prepared by coating with 50μL of HSV-1 McKrae infected cell lysate (20 μg/ml) at 4’C overnight. Then plates were washed and blocked with 5% non-fat dry milk in PBS for 2 hours at room temperature. Then 1:100 diluted serum from each group of mice were added to each well and incubated for 2 hours at room temperature, followed by incubation with Goat Anti-Mouse HRP conjugated antibodies. The TMB substrate solution was used to develop color reaction and optical density of each well will be quantified with a plate reader.
High protein binding plates
Manufactured by Corning
High protein binding plates are designed to efficiently bind and capture proteins from samples. They provide a stable and reproducible surface for protein adsorption, enabling effective analysis and purification.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated streptavidin, by Merck Group (2 mentions)
O phenylenediamine dihydrochloride, by Merck Group (2 mentions)
Spectramax m2, by Molecular Devices (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
5 protocols using high protein binding plates
1
Measuring Antigen-Specific Antibody Isotypes
2
RVFV IgG Antibody Detection ELISA
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To test for evidence of past RVFV infection, serum specimens were screened for the presence of anti-RVF IgG via ELISA [4 (link),7 (link)]. Briefly, high protein binding plates (Corning) were coated with RVFV variant rMP-12 viral antigens prepared in Vero cell lysates, and blocked in 5% non-fat dairy milk. Serum samples were diluted 1:100 in PBS/5% milk solution and allowed to absorb for 1 h at 37°C. After washing, a HRP-conjugated secondary anti-human IgG antibody was applied, again in the milk solution at a 1:2000 dilution. Plates were incubated at 37°C for 1 h then developed using a TMB substrate (Thermo) and absorbance was read at 405nm. Each sample was run in duplicate, and OD values were normalized to background values from wells coated with uninfected Vero cell lysate and averaged. Samples were considered positive with OD values greater than the mean + 2 SD for pooled control sera obtained from unexposed North Americans.
3
Measurement of Anti-Salmonella IgG2c Antibodies
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Blood was collected retro-orbitally from malaria-infected or control mice. Sera were prepared by centrifugation. High-protein binding plates (Corning, Tewksbury, MA) were coated overnight with heat-killed Salmonella diluted in 0.1M NaHCO3. After incubation in 10% FBS/PBS for one hour at 37°C, the plates were washed twice with PBS/0.05% Tween 20, and serum samples were added in serial dilutions in 10% FBS/PBS. After incubation for two hours at 37°C, the plates were washed four times before the addition of biotin-conjugated anti-mouse IgG2c antibody (BD Bioscience). After incubation for one hour at 37°C, the plates were washed six times and incubated for one hour at 37°C with HRP-conjugated streptavidin (Sigma-Aldrich). The plates were then washed eight times and an HRP substrate (O-phenylenediamine dihydrochloride, Sigma-Aldrich) was used to develop the plates. After sufficient color-change was observed, the reaction was stopped by adding 100μL 1M H2SO4. The plates were analyzed using a spectrophotometer (SpectraMax M2, Molecular Devices, Sunnyvale, CA).
4
Comparing SARS-CoV-2 Spike and Influenza HA Vaccines
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Mice were s.c. immunized with alum-adsorbed recombinant HA (10 µg) or recombinant full-length SARS-CoV-2 spike protein (5 µg) with TLR7–alum or TLR7-NP (equivalent gardiquimod dose, 20 µg) in 100 µl PBS at tail base at week 0 and boosted at week 3. Sera were collected by face bleeding 2 weeks following either priming or boosting for enzyme-linked immunosorbent assay (ELISA) assays to measure anti-HA titres or anti-SARS-CoV-2 titres. High protein binding plates (Costar) were directly coated with the antigen of interest (2 µg ml−1, 50 µl) overnight. The plates were blocked with the blocking buffer for 1 h, washed and cultured with diluted mouse sera (1:500 or 1:2,500) for 2 h. Horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibodies to IgG (SouthernBiotech, 1031-05, 1:4,000) was used to detect bound antibodies. Plates were developed with TMB substrate solution (Thermo Scientific), quenched with sulfuric acid and read at 450 nm with a microplate reader (Bio-Rad). In all ELISA assays, unvaccinated mouse serum was used as negative control.
5
Measuring Ovalbumin-Specific Antibodies by ELISA
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