A Competitive ELISA (Enzyme-Linked Immunosorbent Assay) is a type of ELISA used to measure the concentration of a specific analyte in a sample by employing a competitive binding mechanism. This method is commonly used when the target analyte (often a small molecule or hapten) is difficult to measure using traditional sandwich ELISA formats due to its small size or low abundance.
Components
- Coated Plate:
- A microplate (usually 96-well) coated with a specific antibody that binds to the analyte or to a hapten.
- Standards:
- Solutions containing known concentrations of the target analyte or a related molecule used to generate a standard curve.
- Sample Diluent:
- A buffer solution used to dilute samples to appropriate concentrations for accurate measurement.
- Detection Antibody:
- An antibody specific to the analyte or hapten, often conjugated to an enzyme like Horseradish Peroxidase (HRP) for detection.
Procedure
- Coating:
- Coat the wells of the microplate with an antibody specific to the analyte or hapten. Incubate to allow binding and then wash away unbound antibody.
- Blocking:
- Block any remaining uncoated surfaces in the wells with a blocking buffer to prevent non-specific binding.
- Addition of Standards and Samples:
- Add standards and samples to the wells. In competitive ELISA, this step involves adding both the sample and a known amount of labeled analyte (or competitor) to the wells.
- Incubation:
- Incubate the plate to allow the analyte in the sample to compete with the labeled analyte for binding to the antibody.
Performance Characteristics
- Sensitivity: High sensitivity, capable of detecting low levels of analytes.
- Precision: Low intra-assay and inter-assay variability, with coefficients of variation (CV) generally below 10%.
- Specificity: Specific to the target analyte or hapten, minimizing cross-reactivity with other substances.
- Linearity: Provides a linear relationship between analyte concentration and optical density over the working range.
Applications
- Hormone Levels: Measurement of hormones in blood or urine.
- Drug Testing: Detection of drugs or their metabolites.
- Toxin Detection: Quantification of toxins in environmental or biological samples.
- Biomarker Analysis: Assessing small molecule biomarkers related to various diseases.
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