Enzyme-linked immunosorbent assay (ELISA) is a plate-based immunoassay used to detect and quantify specific proteins, antibodies, hormones, and other molecules. It combines the specificity of antibodies with the sensitivity of enzyme-based detection.
How ELISA Works
- Coating
A microtiter plate is coated with an antigen or capture antibody. The target molecule binds to the surface through passive adsorption. The plate is incubated overnight and then washed to remove unbound material.
- Blocking
A blocking solution containing an irrelevant protein such as BSA or casein is added to the wells. This covers any remaining binding sites on the plate surface, preventing non-specific binding in subsequent steps.
- Detection Antibody
A detection antibody conjugated to an enzyme—most commonly horseradish peroxidase (HRP) or alkaline phosphatase (AP)—is added. In a sandwich ELISA, a primary antibody binds the target, and an enzyme-linked secondary antibody binds the primary.
- Signal Generation
A substrate for the enzyme is added. The enzyme converts the substrate into a colored, fluorescent, or luminescent product. The reaction is allowed to proceed for a set time and then stopped.
- Measurement
The signal is measured using a plate reader. The intensity of the signal is proportional to the amount of target molecule in the sample. A standard curve using known concentrations allows quantification.
- ELISA Formats
- Direct ELISA: Antigen is coated, and an enzyme-linked antibody detects it directly.
- Indirect ELISA: Antigen is coated, a primary antibody binds, and an enzyme-linked secondary antibody detects it.
- Sandwich ELISA: A capture antibody is coated, the antigen binds, and a detection antibody completes the sandwich.
- Competitive ELISA: Signal decreases as target concentration increases.
Practical Sandwich ELISA Protocol
Coat a 96-well high-binding microtiter plate with 100 µL/well of capture antibody diluted in coating buffer (0.1 M carbonate-bicarbonate buffer pH 9.6) at a concentration of 1–5 µg/mL. Incubate overnight at 4°C. Wash the plate 3 times with 300 µL/well of PBST (PBS + 0.05% Tween-20) using a plate washer or multichannel pipette. Block each well with 200 µL of blocking buffer (PBST containing 3% BSA or 5% non-fat dry milk) for 1 hour at room temperature. Wash 3 times. Add 100 µL/well of standards (serial dilutions of the target protein, typically 7 concentrations ranging from 1000 pg/mL to 15 pg/mL) and samples, each in duplicate. Incubate for 2 hours at room temperature. Wash 5 times. Add 100 µL/well of detection antibody (biotinylated or directly enzyme-conjugated) at the optimized concentration (typically 0.5–2 µg/mL). Incubate for 1 hour at room temperature. Wash 5 times. If using a biotinylated detection antibody, add 100 µL/well of streptavidin-HRP (1:2000) for 30 minutes and wash 5 times. Add 100 µL/well of TMB substrate solution and incubate in the dark for 15–30 minutes. Stop the reaction with 50 µL/well of 2N H2SO4. Measure absorbance at 450 nm with a plate reader within 30 minutes. Generate a standard curve by plotting absorbance vs. log concentration and fit a 4-parameter logistic (4PL) or 5-parameter logistic model. Calculate sample concentrations by interpolating from the standard curve. Acceptable curves have an R² > 0.98 and back-calculated standard concentrations within 70–130% of expected values.
Real-World Application
A sandwich ELISA for human TNFα in patient serum uses a monoclonal capture antibody and a polyclonal detection antibody. The assay has a detection range of 15–1000 pg/mL with a limit of quantification of 20 pg/mL. Serum from rheumatoid arthritis patients shows TNFα levels of 85 ± 32 pg/mL (n = 24) compared to 12 ± 7 pg/mL in healthy controls, confirming TNFα as a biomarker of inflammation and supporting anti-TNFα therapy decisions.
