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Protocol Overview
Sandwich ELISA is the most widely used immunoassay format in clinical diagnostics and biopharmaceutical development. The assay captures the target analyte between two antibodies — a solid-phase capture antibody and a solution-phase detection antibody — then detects the complex through an enzyme-conjugated reagent and substrate reaction.
This protocol provides a general framework for sandwich ELISA. Specific parameters (antibody concentrations, incubation times, substrate selection) should be optimized for your specific antibody pair and analyte. Total assay time: 4–6 hours (or overnight with coating step).
Materials Required
| Category | Item | Durastab Product |
|---|---|---|
| Solid Phase | 96-well high-binding microwell plate | UPST (Ultra Plate Sealer) / PFPS (Protein-free Plate Sealer) |
| Coating Buffer | 0.05 M Carbonate-Bicarbonate, pH 9.6 | 10CB (Coating Buffer 10× CB) |
| Blocking Buffer | High-performance blocking solution | UTBS (Ultra Blocking Solution) / CABS (Casein Blocking Solution) / PFBS (Protein-free Blocking Solution) |
| Sample Diluent | Protein-stabilized diluent with interference reduction | NEPB (Noise Eliminator PBS) / NETR (Noise Eliminator TRIS) / NEPF (Protein-free Noise Eliminator) |
| Antibody Stabilizer | For detection antibody working solution | ABPB (Antibody Stabilizer PBS) / ABTR (Antibody Stabilizer TRIS) |
| Conjugate Stabilizer | For HRP/AP conjugate working solution | HRST (HRP Stabilizer) / HRNE (HRP Stabilizer NE) |
| Wash Buffer | PBS-T (0.05% Tween-20) | PBST-10 (PBS-T 10X) / TBST-10 (TBS-T 10X) |
| Substrate | TMB one-component substrate | TMST (TMB One Component ELISA Substrate) / TMHS (TMB High Sensitive) / TMSS (TMB Super Sensitive) |
| Stop Solution | Acid stop solution for TMB | STTY (TMB Stop Solution, yellow, 450 nm) / STTB (TMB Stop Solution Blue, 650 nm) |
Dilute the capture antibody to the optimal concentration in carbonate-bicarbonate coating buffer (pH 9.6). Add 100 μL per well to the microwell plate. Seal the plate and incubate to allow passive adsorption of the antibody onto the polystyrene surface.
• Antibody concentration: 1–10 μg/mL (optimize by checkerboard titration)
• Volume: 100 μL/well
• Buffer: 0.05 M Carbonate-Bicarbonate, pH 9.6
• Incubation: Overnight at 4°C or 2 h at 37°C
• Seal plate with adhesive film to prevent evaporation
Avoid using outer wells (row A/H, column 1/12) for critical samples — they are more susceptible to edge effect caused by evaporation. Fill outer wells with coating buffer or water as a humidity buffer.
After coating, aspirate the coating buffer and add blocking buffer to saturate all remaining protein-binding sites on the microwell surface. This step is critical for preventing non-specific binding of detection reagents and sample proteins.
• Volume: 200–300 μL/well
• Incubation: 1–2 h at 37°C or overnight at 4°C
• After incubation: Aspirate and blot dry (do not wash)
Add diluted samples and standards to the blocked wells. The target analyte in the sample binds to the immobilized capture antibody. Include a standard curve (minimum 6 concentrations, in duplicate or triplicate) and appropriate controls.
Add the detection antibody diluted in antibody diluent. The detection antibody binds a different epitope on the captured analyte, completing the "sandwich" complex.
Add the enzyme-conjugated secondary reagent (e.g., HRP-streptavidin for biotinylated detection antibodies, or HRP-anti-IgG for indirect detection). The conjugate binds the detection antibody, and the enzyme will convert the substrate into a measurable signal in the next step.
• Volume: 100 μL/well
• Diluent: Durastab HRST (HRP Stabilizer) or HRNE (HRP Stabilizer NE, with Noise Eliminator)
• Dilution: Optimize (typical range: 1:2,000 – 1:100,000)
• Incubation: 30–60 min at 37°C, protected from light
• Wash: 5 × 300 μL PBS-T with 30 s soak (7 washes for high-sensitivity assays)
Add TMB substrate solution to each well. The HRP enzyme converts TMB into a blue-colored product. The intensity of color is proportional to the amount of analyte captured. Protect the plate from light during this step.
• Volume: 100 μL/well
• Substrate: Durastab TMST (TMB One Component ELISA Substrate, standard) / TMHS (TMB High Sensitive) / TMSS (TMB Super Sensitive)
• Incubation: 10–30 min at room temperature, protected from light
• Monitor color development: stop when highest standard reaches OD 1.5–2.5
Verify substrate quality before use: blank OD at 450 nm should be
Add stop solution to terminate the enzyme-substrate reaction. The color changes from blue to yellow (for TMB with acid stop). Read the plate at the appropriate wavelength within the recommended time window.
• Volume: 50–100 μL/well (add to all wells in the same order and at the same rate)
• Stop solution: Durastab STTY (TMB Stop Solution, yellow, 450 nm) / STTB (TMB Stop Solution Blue, 650 nm)
• Read wavelength: 450 nm (reference: 620–650 nm optional)
• Read within 30 minutes of stop solution addition
Optimization Tips
Checkerboard Titration
Before running the full protocol, perform a checkerboard titration to identify the optimal concentrations for both the capture antibody and the conjugate. Test at least 3 capture antibody concentrations (1, 5, 10 μg/mL) against 5 conjugate dilutions (1:2,000 to 1:100,000). Select the combination that provides the highest signal-to-noise ratio — not the highest absolute signal.
Wash Optimization
If background is elevated, increase wash stringency: more cycles (5→7), higher volume (250→300 μL), longer soak time (15→30 s), or higher Tween-20 concentration (0.05%→0.1%). If signal is weak, reduce wash stringency to preserve bound analyte.
Substrate Selection
Choose the substrate grade based on your sensitivity requirements:
- TMST (TMB One Component ELISA Substrate) — Standard sensitivity; routine clinical assays
- TMHS (TMB High Sensitive One Component ELISA Substrate) — High sensitivity; up to 3× signal enhancement
- TMSS (TMB Super Sensitive One Component ELISA Substrate) — Super sensitive; 2–10 min development, ultra-low LOD
- TMBR (TMB Broad Range One Component ELISA Substrate) — Broad range; extended dynamic range for wide-concentration analytes
Blocking:UTBS (Ultra Blocking Solution) / CABS (Casein Blocking Solution) / PFBS (Protein-free Blocking Solution)
Diluent:NEPB (Noise Eliminator PBS) / NETR (Noise Eliminator TRIS) / NEPF (Protein-free Noise Eliminator)
Antibody Stabilizer:ABPB (Antibody Stabilizer PBS) / ABTR (Antibody Stabilizer TRIS) / ABPF (Antibody Stabilizer Protein-free)
Conjugate Stabilizer:HRST (HRP Stabilizer) / HRNE (HRP Stabilizer NE) / HRPF (Protein-free HRP Stabilizer)
Substrate:TMST (TMB One Component ELISA Substrate) / TMHS (TMB High Sensitive) / TMSS (TMB Super Sensitive) / TMBR (TMB Broad Range)
Stop Solution:STTY (TMB Stop Solution, yellow, 450 nm) / STTB (TMB Stop Solution Blue, 650 nm)
Noise Eliminator:NEPL (Noise Eliminator Plus) / NEPF (Protein-free Noise Eliminator)
Heterophilic Blocker:HBR-001 (Heterophilic Antibody Blocker) / HBR-007 (Heterophilic Antibody Blocker)
Frequently Asked Questions
What is the sandwich ELISA principle?
Sandwich ELISA uses two antibodies that recognize different epitopes on the same target analyte. The capture antibody is immobilized on a solid phase (microwell plate), and the detection antibody binds the analyte after it has been captured. An enzyme-conjugated secondary reagent then binds the detection antibody, and addition of a substrate produces a measurable signal proportional to the analyte concentration. This format provides high specificity and sensitivity compared to direct or competitive ELISA.
How long does a sandwich ELISA take?
A standard sandwich ELISA typically takes 4–6 hours from start to finish, including: coating (overnight or 2h at 37°C), blocking (1h at 37°C), sample incubation (1–2h), detection antibody (1h), conjugate (30–60 min), substrate development (10–30 min), and stop/read. Accelerated protocols using pre-coated plates can reduce total time to 2–3 hours.
What is the optimal coating concentration for ELISA?
The optimal coating concentration depends on the antibody affinity and plate binding capacity. A typical starting range is 1–10 μg/mL in carbonate-bicarbonate buffer (pH 9.6) or PBS (pH 7.4). Use checkerboard titration to determine the minimum concentration that provides maximum signal with minimum background. Coating conditions: 100 μL/well, overnight at 4°C or 2 h at 37°C.
Why is blocking necessary in ELISA?
Blocking saturates all unoccupied protein-binding sites on the microwell surface after antibody coating. Without blocking, detection antibodies, conjugates, and sample proteins will adsorb non-specifically to the plate surface, generating elevated background signal. Use Durastab UTBS (Ultra Blocking Solution) for general applications, CABS (Casein Blocking Solution) for high-sensitivity assays, or PFBS (Protein-free Blocking Solution) for animal-component-free workflows.
How do I optimize the substrate incubation time?
Substrate incubation time depends on the substrate sensitivity and analyte concentration. For standard TMB substrate TMST (TMB One Component ELISA Substrate), 15–30 minutes is typical. For high-sensitivity substrates TMHS (TMB High Sensitive) and TMSS (TMB Super Sensitive), development may be complete in 2–10 minutes. Monitor color development visually and stop the reaction when the highest standard reaches the desired OD range (typically 1.5–2.5 OD at 450 nm). Over-development increases background; under-development reduces sensitivity.
What controls should I include in every ELISA plate?
Every ELISA plate should include: (1) Blank wells — coating buffer only, no sample, no conjugate; (2) Negative control — matrix without analyte; (3) Positive control — known analyte concentration; (4) Standard curve — minimum 6 concentrations in duplicate or triplicate; (5) Sample wells — in duplicate minimum. These controls allow you to identify the source of any issues (high background, weak signal, plate defects).
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