Enzyme-Linked Immunosorbent Assay (ELISA)

The Enzyme-Linked Immunosorbent Assay (ELISA) is a highly sensitive and specific immunoassay technique. Its core principle relies on the specific binding between an antigen and an antibody, coupled with an enzyme-catalyzed chromogenic reaction, to qualitatively or quantitatively detect minute amounts of target substance (antigen or antibody) in a sample.
Technical Principle: ELISA technology depends on the interaction between an antigen (the target protein) and a primary antibody directed against it, with confirmation of the antigen's presence achieved via an enzyme-linked antibody catalyzing an added substrate. In ELISA, the liquid sample is added to a fixed solid phase within a reaction chamber or microplate well. After adding the test sample, the target is specifically captured; an enzyme-labeled detection antibody is then added, forming a "capture antibody-target-enzyme-labeled antibody" complex. After washing away unbound components, substrate is added, and the enzyme catalyzes a color change. The color intensity is proportional to the target concentration. Results can be assessed qualitatively by visual inspection or quantified using a microplate reader^[1][2][3][4].
Development and Application: Due to its advantages of high sensitivity, high throughput, relatively simple operation, and low cost, ELISA developed and evolved rapidly. Early direct ELISA methods were optimized, leading to various detection formats like indirect, sandwich, and competitive ELISA, significantly improving detection sensitivity and application scope^{[1][2][3][4][5]}. ELISA is widely used in biomedical research and drug development. It can monitor drug concentrations or neutralizing antibody levels in blood, quantitatively analyze concentrations of biomolecules like cytokines and signaling proteins in cell culture supernatants or body fluids, thereby revealing mechanisms of biological activity and drug targets.

ELISA, based on the specific binding of antigens and antibodies, uses enzyme-labeled antibodies to catalyze a substrate-producing chromogenic reaction for quantitative analysis of target proteins. It typically involves steps: coating, blocking, incubation, detection, and result analysis. Procedures for different ELISA types are described below.

A. Direct ELISA

1.Coating:
(1) Coat with Antigen: Add 50-100 μL of antigen at a predetermined concentration to each well. Seal the plate and incubate overnight at 4°C.
(2) Washing: Wash the plate three times using an ELISA plate washer with 0.05% PBST buffer. Fill and empty wells by aspiration or inverting to remove solution.
2.Blocking:
(3) Blocking: Block the plate with 100-200 μL/well of PBS containing 3% BSA. Seal the plate and incubate at room temperature for 1 hour.
(4) Washing: Wash as in step (2).
3.Incubation:
(5) Add Antibody Conjugate Solution: Add the antibody conjugate solution (Ab-HRP), using the same volume as in step (1), diluted in 0.05% PBST containing 0.5% BSA with two-fold serial dilutions (at least three dilutions). Perform each dilution in triplicate. Seal the plate and incubate at room temperature for 1 hour.
(6) Washing: Remove the solution and wash the plate six times using the ELISA plate washer with 0.05% PBST buffer.
(7) Development: Add TMB substrate solution, using the same volume as in step (1). Incubate at room temperature for 7 minutes or until the desired color intensity is reached (a clear gradient should be visible in the standards).
(8) Stop Reaction: Stop the reaction by adding 50 μL of 2 M H2SO4 solution to each well.
4.Detection and Analysis:
(9) Read Plate: Measure the absorbance using an ELISA plate reader with appropriate hardware.
(10) Data Analysis

B. Indirect ELISA

1.Coating:
(1) Coat with Antigen: Add 50-100 μL of antigen at a predetermined concentration to each well. Seal the plate and incubate overnight at 4°C.
(2) Washing: Wash the plate three times using an ELISA plate washer with 0.05% PBST buffer. Fill and empty wells by aspiration or inverting to remove solution.
2.Blocking:
(3) Blocking: Block the plate with 100-200 μL/well of PBS containing 3% BSA. Seal the plate and incubate at room temperature for 1 hour.
(4) Washing: Wash as in step (2).
3.Incubation:
(5) Add Primary Antibody: Add the primary antibody solution, using the same volume as in step (1), diluted in 0.05% PBST containing 0.5% BSA with two-fold serial dilutions (at least three dilutions). Perform each dilution in triplicate. Seal the plate and incubate at room temperature for 1 hour.
(6) Washing: Wash as in step (2).
(7) Add Secondary Antibody: Add the secondary antibody solution, e.g., Goat Anti-Human IgG H&L (HRP), diluted in 0.05% PBST containing 0.5% BSA, using the same volume as in step (1). Seal the plate and incubate at room temperature for 1 hour.
(8) Washing: Remove the solution and wash the plate six times using the ELISA plate washer with 0.05% PBST buffer.
(9) Development: Add TMB substrate solution, using the same volume as in step (1). Incubate at room temperature for 7 minutes or until the desired color intensity is reached (a clear gradient should be visible in the standards).
(10) Stop Reaction: Stop the reaction by adding 50 μL of 2 M H₂SO₄ solution to each well.
4.Detection and Analysis:
(11) Read Plate: Measure the absorbance using an ELISA plate reader with appropriate hardware.
(12) Data Analysis

C. Sandwich ELISA

1.Coating:
(1) Coat with Capture Antibody: Add 50-100 μL of capture antibody at a predetermined concentration to each well. Seal the plate and incubate overnight at 4°C or for 2 hours at room temperature.
(2) Washing: Wash the plate three times in an ELISA plate washer with 0.05% PBST buffer. Fill and empty wells by aspiration or inverting to remove solution.
2.Blocking:
(3) Blocking: Block the plate with PBS containing 3% BSA (100-200 μL/well). Seal the plate and incubate at room temperature for 1 hour.
(4) Washing: Wash as in step (2).
3.Incubation:
(5) Add Standards and Samples: Add 50-100 μL of standards and samples to each well, using the same volume as in step (1), diluted in 0.05% PBST containing 0.5% BSA with two-fold serial dilutions (at least three dilutions). Perform each dilution in triplicate. Seal the plate and incubate at room temperature for 2 hours.
(6) Washing: Remove the solution and wash the plate six times in the ELISA plate washer with 0.05% PBST buffer.
(7) Add Primary Antibody: Add another specific detection antibody targeting a different epitope on the target antigen, diluted in 0.05% PBST containing 0.5% BSA, 50-100 μL/well, using the same volume as in step (1). Seal the plate and incubate at room temperature for 1 hour.
(8) Add Secondary Antibody: Add the secondary antibody solution, e.g., Goat Anti-Human IgG H&L (HRP), diluted in 0.05% PBST containing 0.5% BSA, 50-100 μL/well, using the same volume as in step (1). Seal the plate and incubate at room temperature for 1 hour.
(9) Washing: Remove the solution and wash the plate six times in the ELISA plate washer with 0.05% PBST buffer.
(10) Development: Add TMB substrate solution, using the same volume as in step (1). Incubate at room temperature for 7 minutes or until the desired color intensity is reached (a clear gradient should be visible in the standards).
(11) Stop Reaction: Stop the reaction by adding 50 μL of 2 M H2SO4 solution to each well.
4.Detection and Analysis:
(12) Read Plate: Measure the absorbance using an ELISA plate reader with appropriate hardware.
(13) Data Analysis

D. Competitive ELISA

1.Coating:
(1) Coat with Antigen/Antibody: Add 50-100 μL of specific antigen or capture antibody at a predetermined concentration to each well. Seal the plate and incubate overnight at 4°C or for 2 hours at room temperature.
(2) Washing: Wash the plate three times in an ELISA plate washer with 0.05% PBST buffer. Fill and empty wells by aspiration or inverting to remove solution.
2.Blocking:
(3) Blocking: Block the plate with PBS containing 3% BSA (100-200 μL/well). Seal the plate and incubate at room temperature for 1 hour.
(4) Washing: Wash as in step (2).
3.Competitive Incubation:
(5) Add Sample/Primary Antibody Mixture: Mix the test sample (or standard) with a predetermined concentration of enzyme-labeled primary antibody (detection antibody) in equal volumes. Add 50-100 μL of this mixture to each well, using the same volume as in step (1). Seal the plate and incubate at room temperature for 1-2 hours.
Note: In this step, free antigen in the sample (or standard) competes with the immobilized antigen/antibody on the plate for binding the limited amount of enzyme-labeled primary antibody.
4.Washing and Development:
(6) Washing: Remove the solution and wash the plate six times in the ELISA plate washer with 0.05% PBST buffer to remove unbound enzyme-labeled primary antibody.
(7) Development: Add TMB substrate solution, using the same volume as in step (1). Incubate at room temperature for 7-15 minutes or until the desired color intensity is reached.
Note: The higher the concentration of the target in the sample, the less enzyme-labeled antibody binds, resulting in weaker final color.
(8) Stop Reaction: Stop the reaction by adding 50 μL of 2 M H2SO4 solution to each well.
5.Detection and Analysis:
(9) Read Plate: Measure the absorbance using an ELISA plate reader with appropriate hardware.
(10) Data Analysis: The absorbance value is inversely proportional to the concentration of the target in the sample. Calculate the concentration of unknown samples using the standard curve.

1. Reagent Temperature Equilibration: Allow all reagents (especially standards, detection antibodies, and enzyme conjugates) to equilibrate to room temperature (approx. 20-30 minutes) before use to minimize well-to-well temperature variation and ensure uniform reaction. TMB substrate solution should be protected from prolonged light exposure.
2. Sample Handling: Clearly identify the sample type (serum, plasma, cell supernatant, tissue homogenate, etc.) and perform appropriate pre-treatment (e.g., centrifugation to remove precipitates). Based on pre-experimental results, dilute samples to the optimal detection range using the recommended diluent.
3. Sample and Reagent Addition: When adding liquids, keep pipette tips close to the well bottom without touching the bottom or walls. Dispense slowly to avoid bubble formation, which can interfere with light path and liquid volume. It is recommended to set up replicates (at least duplicates) for standards and important samples to assess experimental reproducibility, take averages, and reduce random error.
4. Incubation: Seal the plate with a plate sealer during every incubation step to prevent evaporation and contamination. Place the reaction plate steadily on a level surface during incubation, avoiding vibration and tilting to ensure uniform reaction. Strictly control incubation time and temperature.
5. Washing: Ensure each well is filled completely with wash buffer (typically 300-350 μL) during each wash step. After adding wash buffer, let it stand for 30-60 seconds to allow unbound substances to fully dissolve and separate before discarding. Flick or snap the plate decisively and blot firmly on absorbent paper. Proceed immediately to the next step after washing; never allow the wells to dry out, as this can deactivate reagents and increase background. If a pause is necessary, it should be after adding the next reagent.
6. Development and Stopping: TMB substrate is light-sensitive; the development step should be performed protected from light. Closely monitor the color gradient in the standard wells to control development time. The order of adding stop solution should be consistent with the order of adding substrate solution to ensure equal reaction time for all wells.
7. Plate Reading: After stopping the reaction, the solution turns from blue to yellow. Reading should be completed within the time specified in the protocol (usually within 30 minutes). Clean the bottom of the plate to remove fingerprints or smudges that could affect data accuracy.