![]() Discard the detection antibody solution and wash the wells three times with the wash buffer.ġ1. Add the detection antibody to the wells and incubate for 1 hour at room temperature.ġ0. Discard the sample solution and wash the wells three times with the wash buffer.ĩ. Incubate for 2 hours at room temperature or overnight at 4☌.Ĩ. Add the standard/sample/competitor antigen mixture to the coated and blocked wells of the microtiter plate. Mix the standard solutions or samples with the competitor antigen in appropriate proportions, and incubate for at least 1 hour at room temperature.ħ. Prepare the samples to be tested in the same standard/sample dilution buffer.Ħ. Prepare a series of standard solutions of the target analyte in the standard/sample dilution buffer, ranging from high to low concentrations.ĥ. Block the wells with the blocking buffer for at least 1 hour at room temperature.Ĥ. Discard the coating buffer and wash the wells three times with the wash buffer.ģ. Incubate overnight at 4☌ or for at least 2 hours at room temperature.Ģ. Prepare the coating buffer and use it to coat the wells of a microtiter plate with the primary antibody against the target analyte. synthetic peptide or recombinant protein)ġ. 2N H2SO4 for TMB substrate or 1% SDS for ABTS substrate) HRP-conjugated secondary antibody against the primary antibody) monoclonal or polyclonal antibody against the target analyte) Here, we provide a comprehensive protocol for conducting a competitive ELISA. The degree of competition between the labeled and unlabeled antigen is directly proportional to the concentration of the unlabeled antigen present in the sample. Competitive ELISA is a technique used to measure the concentration of a specific antigen in a sample by assessing its ability to compete with a labeled antigen for binding to a limited amount of immobilized capture antibody. ![]()
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