ELISA was performed as described previously.10 (link) Briefly, 96-well high-binding polystyrene plates (Greiner Bio-One, Austria) were coated with recombinant proteins at 2.5 μg mL−1 in carbonate-bicarbonate buffer, pH 9.6, at 4°C overnight. The wells were then blocked with 5% skim milk. Different sera dilutions were added to the wells and allowed to interact at 37°C for 1 h. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (catalog no. 1030-05), IgG1 (catalog no. 1070-05), or IgG2a (catalog no. 1080-05) antibody at 1:5,000 or 1:3,000 was added, and the plates were incubated at 37°C for 1 h. The goat anti-hamster IgG HRP (catalog no. 6060-05) was used at a dilution of 1:10,000. At each step, plates were stringently washed with 0.1% PBST. The assay was developed using OPD substrate for 5–10 min in the dark. The ODs were read at 492 nm in a microplate reader (Tecan, Switzerland) after stopping the reaction with 3 M H2SO4. Additionally, IgA response in sera of immunized was mice was also determined by using goat anti-mouse IgA HRP (catalog no. 1040-05) at 1:3,000. All HRP-conjugated secondary antibodies used were from Southern Biotech, USA.
96 well high binding polystyrene plates
Manufactured by Greiner
Sourced in Austria
96-well high-binding polystyrene plates are a type of laboratory equipment used for various applications. These plates feature a high-binding surface made of polystyrene, designed to facilitate the adsorption of biomolecules, such as proteins, peptides, or antibodies, onto the well surfaces. The 96-well format provides a standardized and efficient platform for various assays, experiments, and screening processes conducted in life science research and diagnostic laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Tecan (3 mentions)
Hrp conjugated secondary antibody, by Southern Biotech (1 mentions)
Hrp conjugated goat anti mouse igg antibody, by Southern Biotech (1 mentions)
Hrp conjugated goat anti mouse igg and igm, by Southern Biotech (1 mentions)
Automated elisa spectrophotometer, by Tecan (1 mentions)
O phenylenediamine dihydrochloride opd, by Merck Group (1 mentions)
4 protocols using 96 well high binding polystyrene plates
1
ELISA Protocol for Antibody Detection
2
Mouse IgG ELISA Quantification
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Total IgG was measured from sera collected at 14 and 21 dpi by ELISAs. Briefly, 96-well high-binding polystyrene plates (Greiner Bio-One, Kremsmünster, Austria) were coated with recombinant proteins at 5 μg/mL in carbonate-bicarbonate buffer, pH 9.6, and incubated at 4 °C overnight. The wells were blocked with 200 μL of 5% skim milk at 37 °C for 1 h and washed with 0.1% PBST. Serum samples were added, incubated at 37 °C for 1 h, and washed as above. HRP-conjugated goat anti-mouse IgG antibody at 1:5000 (Southern Biotech, USA) was added and incubated at 37 °C for 1 h. Finally, colour was developed with 100 μL of freshly prepared OPD substrate in phosphate-citrate buffer (pH 5.0) containing H2O2 at room temperature for 5–10 min in the dark. The reaction was stopped by adding 50 μL of 3 M H2SO4, and the optical density (OD) was measured at 492 nm in a microplate reader (Tecan, Zürich, Switzerland).
3
Antibody Titer Determination by ELISA
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End-point antibody titers in mice sera collected at weeks 3 and 5 post-immunization was determined by ELISA. Briefly, 96-well high-binding polystyrene plates (Greiner Bio-One, Austria) were coated with recombinant proteins at 2.5 µg mL−1 in carbonate-bicarbonate buffer, pH 9.6 at 4 °C overnight. The wells were then blocked at 37 °C for 1 h with 200 µL 5% skim milk and washed three times with 0.1% PBST. Serial dilutions of 100 µL sera were added to the wells, and plates were incubated at 37 °C for 1 h and then washed as above. HRP-conjugated goat anti-mouse IgG, IgG1 or IgG2a antibody at 1:5000 or 1:3000 (Southern Biotech, USA) was added and the plates were incubated at 37 °C for 1 h. Finally, following stringent washing, the assay was developed with 100 µL of freshly prepared OPD substrate in phosphate-citrate buffer (pH 5.0) containing H2O2 at room temperature for 5–10 min in the dark. The optical densities (OD) were read at 492 nm in a microplate reader (Tecan, Switzerland) after stopping the reaction with 50 μL of 3 M H2SO4. The highest serum dilution yielding an absorbance 2.1-fold higher than negative control was recorded as the endpoint titer [40] (link).
4
Quantifying Antigen-Specific Antibody Levels
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Antigen-specific immunoglobulin (Ig) G and IgM levels were measured by indirect ELISA using serum samples collected on weeks 0, 1, 2, 4, 6, and 8 post-primary vaccination. Briefly, purified antigens were coated (500 ng/well) on 96-well high-binding polystyrene plates (Greiner Bio-One, Kremsmuster, Austria) and incubated at 4 °C overnight. The following day, wells were blocked with 5% skim milk (BD Difco) for 1 h at room temperature and washed three times with 0.1% phosphate-buffered saline with tween 20 (PBSt). Next, wells were incubated with serum samples (1:50) for 1 h at 37 °C, and plates were washed three times with PBSt. HRP-conjugated goat anti-mouse IgG and IgM (Southern biotech, AL, USA) secondary antibodies at 1:3000 dilution were added, and the plates were incubated at 37 °C for 1 h. After washing three times with PBSt, Color development was achieved with the prepared O-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich) substrate in phosphate-citrate buffer containing H2O2 at room temperature in the dark. Optical density (OD) was measured at 492 nm using an automated ELISA spectrophotometer (Tecan, Groedig, Austria).
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