ELISA Protocol: Step-by-Step Guide for Sandwich and Indirect ELISA

The enzyme-linked immunosorbent assay (ELISA) is one of the most widely used techniques in biomedical research for detecting and quantifying proteins, antibodies, hormones, and other analytes in complex biological samples. First described by Engvall and Perlmann in 1971, ELISA combines the specificity of antibody-antigen recognition with the sensitivity of enzymatic signal amplification — routinely achieving detection limits in the low picogram-per-milliliter range.

This guide provides optimized protocols for the two most commonly used ELISA formats: sandwich ELISA (the gold standard for quantifying soluble antigens) and indirect ELISA (used for detecting antibodies in serum or assessing antibody titer).

1. ELISA Formats at a Glance

2. Protocol A: Sandwich ELISA

Sandwich ELISA is the most widely used format for quantifying soluble proteins (cytokines, growth factors, hormones, drug molecules) in biological fluids. It uses a matched antibody pair: one capture antibody immobilized on the plate and one detection antibody that recognizes a different epitope on the same target.

Materials

96-well ELISA microplate (high-binding polystyrene, flat-bottom), coating buffer (PBS pH 7.4 or carbonate/bicarbonate buffer pH 9.6), blocking buffer (1–3% BSA in PBS or dedicated ELISA blocking buffer), wash buffer (PBS + 0.05% Tween 20, PBST), capture antibody, detection antibody (biotinylated or HRP-conjugated), streptavidin-HRP (if using biotinylated detection Ab), TMB substrate solution, stop solution (0.16–2 M H₂SO₄), recombinant protein standard, microplate reader (450 nm).

Step-by-Step Protocol

Step 1: Coat the Plate with Capture Antibody

Dilute the capture antibody to 1–10 µg/mL in coating buffer (PBS pH 7.4 or carbonate buffer pH 9.6). Add 100 µL per well. Seal the plate and incubate overnight at 4°C.

Alternatively, incubate for 1–2 hours at 37°C for faster turnaround, though overnight coating generally yields more consistent results.

Step 2: Wash & Block

Aspirate the coating solution and wash 3× with 300 µL PBST per well. Add 200–300 µL blocking buffer (1–3% BSA in PBS) to each well. Incubate for 1 hour at room temperature.

Blocking prevents non-specific protein adsorption to unoccupied plate surfaces. Wash 3× with PBST after blocking.

Step 3: Add Samples & Standards

Prepare a 7–8-point serial dilution of the recombinant protein standard in reagent diluent (1% BSA in PBS). Include a blank (diluent only). Dilute samples as needed.

Add 100 µL of each standard or sample per well in duplicate. Seal the plate and incubate for 2 hours at room temperature (or overnight at 4°C for low-abundance analytes).

Step 4: Detection Antibody

Wash 3–5× with PBST. Add 100 µL of detection antibody (biotinylated or HRP-conjugated) diluted in reagent diluent at the manufacturer's recommended concentration (typically 0.25–2 µg/mL). Incubate for 1–2 hours at room temperature.

Step 5: Enzyme Conjugate (if using biotinylated detection Ab)

Wash 3–5× with PBST. Add 100 µL streptavidin-HRP (diluted per manufacturer's instructions, typically 1:200–1:2000) to each well. Incubate for 20–30 minutes at room temperature. If the detection antibody is directly HRP-conjugated, skip this step.

Step 6: Substrate Development

Wash 5× with PBST (extra washes reduce background at this stage). Add 100 µL TMB substrate solution to each well. Incubate for 15–30 minutes at room temperature in the dark (cover with foil).

TMB produces a blue color proportional to the amount of bound HRP. When the top standard wells reach an appropriate color intensity (OD around 1.0–2.0), add 50–100 µL of stop solution (2 M H₂SO₄). The color changes from blue to yellow.

Step 7: Read & Analyze

Read the plate immediately at 450 nm on a microplate reader. Subtract blank OD values. Plot the standard curve and interpolate sample concentrations (see Section 4).

3. Protocol B: Indirect ELISA

Indirect ELISA is used primarily to detect and quantify antibodies in a sample — for example, measuring serum IgG titer against a specific antigen after immunization, or screening hybridoma supernatants.

Step-by-Step Protocol

1. Coat: Dilute the target antigen (recombinant protein or peptide) to 1–10 µg/mL in coating buffer. Add 100 µL per well. Incubate overnight at 4°C.

2. Block: Wash 3× PBST. Add 200 µL blocking buffer (1–3% BSA in PBS). Incubate 1 h at RT.

3. Sample incubation: Wash 3×. Add 100 µL diluted serum or sample (serial dilutions for titer determination). Incubate 1–2 h at RT.

4. Secondary antibody: Wash 3–5×. Add 100 µL HRP-conjugated secondary antibody (e.g., goat anti-mouse IgG-HRP for mouse sera). Incubate 1 h at RT.

5. Substrate: Wash 5×. Add 100 µL TMB. Develop 15–30 min in the dark.

6. Stop & Read: Add stop solution. Read at 450 nm.

4. Standard Curve Construction & Data Analysis

A properly constructed standard curve is essential for converting raw OD values to analyte concentrations.

1. Prepare a 2-fold serial dilution series of the recombinant protein standard (typically 7–8 points + blank).

2. Plot absorbance (Y-axis, linear) vs. concentration (X-axis, log scale).

3. Fit the curve using a 4-parameter logistic (4PL) regression — the recommended model for sigmoidal ELISA curves. Most plate reader software supports 4PL fitting.

4. Interpolate sample concentrations from the standard curve. Multiply by the dilution factor to obtain the original sample concentration.

5. Optimization Tips

6. Frequently Asked Questions

Q: When should I use sandwich ELISA vs. indirect ELISA?

Use sandwich ELISA when you want to quantify a specific soluble antigen (e.g., cytokine concentration in cell culture supernatant, drug level in serum). It requires a matched antibody pair but provides the highest specificity and sensitivity. Use indirect ELISA when you want to detect or measure antibodies in a sample (e.g., serum antibody titer after vaccination, or screening hybridoma clones for antibody production). In indirect ELISA, the antigen is coated on the plate and the sample antibody is the analyte.

Q: Why is my standard curve flat at the top?

A plateau at the upper end indicates substrate saturation or capture antibody saturation. Common causes: over-development of TMB substrate (stop the reaction sooner); capture antibody concentration too low to bind all available antigen at high concentrations; or the enzyme conjugate concentration is insufficient. If reducing development time does not fix it, increase the coating antibody concentration or reduce the top standard concentration.

Q: Can I use the same antibody as both capture and detection?

Only if the target antigen has multiple copies of the same epitope (e.g., a homodimer or a polymer). For most soluble monomeric proteins, you need two antibodies recognizing different epitopes. Using the same antibody for both capture and detection will not form a sandwich because the capture antibody already occupies the epitope.

Q: What is the difference between HRP and AP detection?

HRP (horseradish peroxidase) with TMB substrate is the most widely used system: it is fast, inexpensive, and produces a strong blue-to-yellow signal. AP (alkaline phosphatase) with pNPP substrate offers a broader linear range and the reaction does not need to be stopped, but it is slower and less sensitive. For most routine applications, HRP/TMB is recommended. AP is useful when working with samples containing endogenous peroxidase that could interfere with HRP-based detection.

Q: How do I troubleshoot high background in my ELISA?

High background in ELISA is most often caused by: inadequate washing (increase wash cycles, especially before substrate); insufficient blocking (try higher BSA concentration or longer blocking time); antibody concentrations too high (titrate down); plate left too long in substrate (monitor development and stop earlier); or cross-reactivity between capture and secondary antibodies. For detailed troubleshooting, see our companion article: ELISA Troubleshooting Guide: Solutions for Common Assay Pitfalls.

References

1. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA): quantitative assay of immunoglobulin G. Immunochemistry. 1971;8(9):871-874. doi: 10.1016/0019-2791(71)90454-x

2. Lequin RM. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem. 2005;51(12):2415-2418. doi: 10.1373/clinchem.2005.051532

3. Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015;72:4-15. doi: 10.1016/j.peptides.2015.04.012

This article is provided for educational purposes only. Protocols should be optimized for each specific application. For technical support, contact order@abinscience.com.