In order to confirm the mapped epitope recognized by MAb 1B3 in H. parasuis, a competitive inhibition ELISA was performed. The 96-well plates were coated with 10 µg/well whole-cell lysates of H. parasuis HS80 strain in 0.1 M NaHCO3 (pH 8.6) at 4°C overnight, and then blocked with 5% skimmed milk diluted in PBS for 1 h at 37°C. Serial dilutions of synthetic peptides were pre-incubated separately with MAb 1B3 (0.5 µg/mL) for 1 h at 37°C. The antibody-peptide mixture was incubated for 20 min at 37°C. The HRP-conjugated goat anti-mouse antibody (1∶5000) was added for 1 h after the plates were washed five times. TMB substrate (Tian Gen, China) was added for colorimetric detection. The results were analyzed using a spectrophotometer at an absorbance of 450 nm.
Tmb substrate
TMB substrate is a commonly used chromogenic substrate in various immunoassays, such as enzyme-linked immunosorbent assay (ELISA). It is a colorless solution that, when catalyzed by the enzyme horseradish peroxidase (HRP), produces a blue-colored product. The intensity of the color change is proportional to the amount of target analyte present in the sample, making it a useful tool for quantitative and qualitative analysis.
7 protocols using tmb substrate
Mapping Antibody Epitope in H. parasuis
In order to confirm the mapped epitope recognized by MAb 1B3 in H. parasuis, a competitive inhibition ELISA was performed. The 96-well plates were coated with 10 µg/well whole-cell lysates of H. parasuis HS80 strain in 0.1 M NaHCO3 (pH 8.6) at 4°C overnight, and then blocked with 5% skimmed milk diluted in PBS for 1 h at 37°C. Serial dilutions of synthetic peptides were pre-incubated separately with MAb 1B3 (0.5 µg/mL) for 1 h at 37°C. The antibody-peptide mixture was incubated for 20 min at 37°C. The HRP-conjugated goat anti-mouse antibody (1∶5000) was added for 1 h after the plates were washed five times. TMB substrate (Tian Gen, China) was added for colorimetric detection. The results were analyzed using a spectrophotometer at an absorbance of 450 nm.
ELISA for H. parasuis Antibody
ELISA for Parasitic Infection Diagnosis
Mxra8-Fc Binding Assay for CHIKV
Enzyme-Linked Immunosorbent Assay for Autoantibody Detection
IgG Antibody Quantification by ELISA
Chikungunya E2 Glycoprotein Epitope Mapping
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