Enzyme-Linked Immunosorbent Assay, commonly known as ELISA, is a widely used laboratory technique designed to detect and quantify various substances, such as proteins, antigens, or antibodies, within biological samples. This plate-based assay relies on specific antibody-antigen interactions to identify and measure target molecules. While several types of ELISA exist, competitive ELISA represents a distinct format employed for particular analytical challenges. This method offers a precise way to determine the concentration of a target substance by observing its interference with a known reaction.
Understanding the Basics
Competitive ELISA operates on the principle of competition for binding sites. The target analyte in a sample competes with a known, labeled version of the same analyte for a limited number of binding sites on an antibody or antigen. A distinguishing characteristic is that the signal generated is inversely proportional to the amount of target analyte in the sample; a higher concentration results in a weaker final signal.
The Step-by-Step Process
Performing a competitive ELISA begins by coating a multi-well plate with either a specific antibody or the target antigen. The sample containing the unknown analyte is then introduced, often pre-incubated with a primary antibody or a labeled competitor. The analyte in the sample competes with a known competitor for binding to the immobilized antibody or antigen. After an incubation period, unbound components are removed through washing steps.
Following washing, an enzyme-conjugated secondary antibody, which binds to the primary antibody, is added. This secondary antibody carries an enzyme, such as horseradish peroxidase (HRP), which facilitates a detectable reaction. After another wash to remove unbound enzyme conjugate, a colorless substrate specific to the enzyme is introduced. The enzyme catalyzes a reaction, leading to a measurable color change or other signal. The intensity of this color development is then measured, providing an indirect indication of the original analyte concentration.
Key Applications
Competitive ELISA is suited for detecting small molecules difficult to analyze with other immunoassays. These small molecules, such as hormones, drugs, pesticides, or environmental contaminants, lack multiple binding sites for sandwich ELISA. This approach allows quantification of molecules with a single epitope. It has broad utility in clinical diagnostics (e.g., hydrocortisone), drug discovery, and screening food or environmental samples. Its ability to measure analytes in complex mixtures like plasma or serum makes it a valuable tool.
Interpreting Your Results
Interpreting competitive ELISA results involves understanding the inverse relationship between the measured signal and analyte concentration. A strong signal, indicated by intense color development, signifies a low concentration of the target analyte. Conversely, a weak signal suggests a high concentration. To quantify unknown samples, a standard curve is generated using known concentrations of the target analyte.
This standard curve plots signal intensity (e.g., optical density) against corresponding known analyte concentrations, often resulting in a sigmoidal curve. By comparing an unknown sample’s signal to this standard curve, its precise analyte concentration can be determined. If samples were diluted before testing, the calculated concentration must be multiplied by the dilution factor to obtain the true concentration.