Complete Sandwich ELISA Protocol

Research will be limited to:
- Assay: Sandwich ELISA
- Format: Microplate based protocol
- Reagents: Defined coating concentration, blocking buffer, primary and secondary antibodies, TMB substrate, stop solution are REQUIRED parameters
- Quantitation: Protocol must include antibody dilutions

Strategy: The user requires a complete, standard sandwich ELISA protocol with specific quantitative parameters (coating concentration, dilutions, and reagent types). I will search for established laboratory protocols (e.g., from Abcam, Thermo Fisher, or R&D Systems) that provide these standard ranges and concentrations to ensure the protocol is technically sound and complete.
Queries:
- standard sandwich ELISA protocol coating concentration blocking buffer antibody dilutions TMB stop solution
- typical capture antibody coating concentration and detection antibody dilution sandwich ELISA
- sandwich ELISA blocking buffer composition and TMB stop solution concentration

🔍 Reading and summarizing 5 new websites...

1. Basic Sandwich ELISA Protocols | Thermo Fisher Scientific - ES
2. Sandwich ELISA protocol - Abcam
3. Sandwich ELISA Protocol - Fortis Life Sciences
4. A Guide to Building a Direct Sandwich ELISA | Proteintech Group
5. [PDF] Sandwich ELISA Protocol - AntibodySystem

Found 4 websites. Summarized content:

Basic Sandwich ELISA Protocols | Thermo Fisher Scientific - ES
Here's a sandwich ELISA protocol based on the provided Thermo Fisher Scientific content, dated March 12, 2026, incorporating your requested parameters:

Assay: Sandwich ELISA (Microplate based)

Protocol (Colorimetric Detection using TMB Substrate)

1. Plate Coating:

• Coating Antibody: Dilute capture antibody in coating buffer (either carbonate or phosphate buffer).
• Coating Concentration: Use a concentration of 1-12 µg/mL for affinity-purified polyclonal or monoclonal antibodies. For polyclonal serum or crude ascites, use 5-15 µg/mL.
• Coat wells with 100 µL/well.
• Incubate: 1 hour at room temperature or overnight (12-18 hours) at 2-8°C.

2. Washing:

• Aspirate and wash once with >300 µL/well of wash buffer (e.g., TBS-Tween or PBS-Tween).
• Invert and tap the plate on absorbent paper to remove excess liquid.

3. Blocking:

• Blocking Buffer: Use a blocking buffer such as SuperBlock (TBS or PBS).
• Add 300 µL/well.
• Incubate: 1 hour at room temperature.
• Aspirate and tap the plate on absorbent paper to remove excess liquid.

4. Standards and Samples:

• Prepare standards and sample dilutions in blocking buffer.
• Add 100 µL of standards and samples (in duplicate) to designated wells.
• Incubate: 1-2 hours at room temperature with gentle shaking (~500 rpm).

5. Washing:

• Aspirate and wash the wells with >300 µL/well of wash buffer.
• Repeat the wash step five times. Invert and tap the plate on absorbent paper to remove excess liquid.

6. Detection Antibody:

• Secondary Antibody Dilutions: Dilute the detection antibody in blocking buffer. Refer to manufacturer’s instructions for specific dilutions.
• For HRP, concentrations range from 20-200 ng/mL for colorimetric. For biotinylated detection antibody, a streptavidin-HRP conjugate is needed.
• Add 100 µL of diluted detection antibody to each well.
• Incubate: 2 hours at room temperature with gentle shaking (~500 rpm).

7. Washing:

• Aspirate and wash the wells with >300 µL/well of wash buffer.
• Repeat the wash step five times. Invert and tap the plate on absorbent paper to remove excess liquid.

8. Streptavidin-HRP (If using biotinylated detection antibody):

• Prepare a working solution of Streptavidin-HRP in blocking buffer at a 1:5,000 dilution.
• Add 100 µL of Streptavidin-HRP solution into each well.
• Incubate: 1 hour at room temperature with gentle continual shaking (~500 rpm).
• Aspirate contents and wash the wells with >300 µL of wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times.

9. Substrate:
* Substrate: TMB substrate solution.
* Add 100 µL of TMB substrate solution to each well.
* Incubate: 30 minutes at room temperature (or until desired color intensity is reached).

10. Stop Solution:

• Stop Solution: Use a stop solution for TMB substrates (e.g. 2N Sulfuric Acid).
• Add 100 µL of stop solution to each well.

11. Measurement:

• Measure absorbance at 450 nm within 30 minutes of adding the stop solution.

12. Data Analysis:

• Calculate results using a log-log or 4-parameter curve fit.

Important Considerations:

• Optimize antibody dilutions for best results. The provided concentrations are starting guidelines.
• Wash buffer is typically PBS or TBS with Tween-20.
• Use affinity-purified antibodies for optimal signal-to-noise ratio.

Additional Notes:

• The protocol can be adapted for chemiluminescent or fluorescent detection with appropriate substrates and readers.
• For chemiluminescent detection, use a luminol-based substrate.
• For fluorescent detection, use QuantaBlu substrate.

Sandwich ELISA protocol - Abcam

Sandwich ELISA Protocol Summary

Here's a Sandwich ELISA protocol based on the provided Abcam website content, addressing the user's request for specific parameters:

1. Plate Coating:

• Coating Concentration: Capture antibody diluted in coating buffer (carbonates e.g., Abcam's ab210899) at a suggested concentration of 1-10 µg/mL.
• Procedure: Add ~50 µL of diluted capture antibody to each well of an uncoated microplate (e.g. Abcam's ab210903). Incubate with gentle agitation for 2 hours at room temperature or overnight at 4°C.

2. Blocking:

• Blocking Buffer: Add 200 µL of blocking buffer (e.g., PBS with 3-5% w/v BSA) to each well.
• Procedure: Block with gentle agitation for 1-2 hours at room temperature, or overnight at 4°C.

3. Sample Incubation:

• Dilute your samples, controls and standards in dilution buffer (e.g., PBS with 3 – 5% w/v BSA) to concentrations within the expected dynamic range of the assay.
• Add 100 µL of diluted samples and standards to the wells and incubate with gentle agitation for 2 hours at room temperature, or 4°C overnight.

4. Primary Antibody (Detection Antibody) Incubation:

• Dilution: Dilute unconjugated or conjugated detection antibody in blocking buffer (e.g., Abcam's ab126587). Optimum dilutions often suggested on the antibody datasheet. If not included, perform serial dilutions to find the appropriate concentration.
• Procedure: Add 100 µL of diluted unconjugated detection antibody to each well and incubate for 2 hours at room temperature or overnight at 4°C. If using a conjugated secondary antibody proceed to secondary antibody incubation. If a conjugated detection antibody is being used, proceed to step 6.

5. Secondary Antibody Incubation (if using an unconjugated primary antibody):
* Add 100 µL of the conjuagted secondary antibody diluted in blocking buffer (e.g., Abcam's ab126587) to each well.
* Incubate for 1 – 2 h at room temperature and proceed to step 6.

6. Washing: Wash each well three times with wash buffer (e.g. Abcam's ab206977 or ab172375) after blocking, sample incubation, primary/detection antibody incubation, and after secondary antibody incubation (if applicable).

7. TMB Substrate:

• Add 50-100 µL of TMB substrate (e.g., Abcam's ab171523) to each well. Incubate with gentle agitation on a plate shaker, as directed by the manufacturer.

8. Stop Solution:

• Add 100 µL of stop solution (e.g., Abcam's ab171529 or ab171531) to each well. Shake the plate for 1 minute to mix.

9. Detection: Read the signal development in a plate reader at the appropriate wavelength.

Sandwich ELISA Protocol - Fortis Life Sciences
Here's a Sandwich ELISA protocol based on the provided information, focusing on the parameters you requested:

Sandwich ELISA Protocol

Assay Type: Sandwich ELISA

Format: Microplate

Reagents and Procedure: (Performed at Room Temperature)

1. Plate Coating:

   • Capture Antibody: Dilute to 2-10 mcg/mL in Coating Buffer (0.05 M Carbonate-Bicarbonate, pH 9.6).
   • Volume: 100 mcL per well.
   • Incubation: 60 minutes.
   • Wash: 3 times with Wash Solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0).

2. Blocking:

   • Blocking Buffer: 50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0
   • Volume: 200 mcL per well.
   • Incubation: 30 minutes.
   • Wash: 3 times with Wash Solution.

3. Standards and Samples:

   • Dilute standards in Sample Diluent (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) to desired concentrations.
   • Dilute samples in Sample Diluent to fall within the standard concentration range.
   • Volume: 100 mcL per well.
   • Incubation: 60 minutes.
   • Wash: 3 times with Wash Solution.

4. HRP-Conjugated Primary Antibody:

   • Dilute HRP-Conjugated Primary Antibody in Sample/Conjugate Diluent
   • Volume: 100 mcL per well.
   • Incubation: 60 minutes.
   • Wash: 5 times with Wash Solution.

5. Enzyme Substrate Reaction:

   • Substrate: TMB (recommended), OPD, or ABTS. Prepare according to manufacturer's instructions.
   • Volume: 100 mcL per well.
   • Incubation: 5-30 minutes (adjust based on color development).

6. Stop Solution:

   • Stop Solution: 100 mcL of 0.18 M H2SO4 per well for TMB. Use manufacturer's recommended stop solution for other substrates (OPD/ABTS).

Additional Reagent Details & Buffers:

• Coating Buffer (0.05 M Carbonate-Bicarbonate, pH 9.6):
  • 3.7 g Sodium Bicarbonate (NaHCO3)
  • 0.64 g Sodium Carbonate (Na2CO3)
  • 1 L of distilled water
• Wash Solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0):
  • Start with TBS (Tris Buffered Saline)
    • 6.06 g Tris Base
    • 8.2 g NaCl
    • 6.0 ml 6 M HCl
    • 1 L of distilled water
    • pH should be 7.2 to 7.8, conductivity should be 14,000 to 16,000
  • Add 5 ml of 10% Tween 20 to 1 L of TBS
• Blocking (Postcoat) Solution (50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0):
  • 1 L of TBS
  • 10 g BSA
• Sample/Conjugate Diluent (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0):
  • 1 L of TBS
  • 10 g of BSA
  • 5 ml of 10% Tween 20
• Stop Solution (0.18 M H2SO4):
  • 10 ml of 18 M H2SO4 stock solution to 1 L distilled water

Plate Type: High Protein Binding Microtiter Plate (i.e. Nunc C bottom Immunoplate 96 well, 446612)

A Guide to Building a Direct Sandwich ELISA | Proteintech Group
Here's a Sandwich ELISA protocol based on the provided text:

Sandwich ELISA Protocol

1. Plate Coating:

• Coat a 96-well microplate (high-binding or medium-binding) with capture antibody at 1-10 µg/mL in carbonate/bicarbonate buffer (pH 9.6).
• Incubate overnight at 4°C.

2. Blocking:

• Remove coating solution.
• Block remaining protein-binding sites with blocking buffer (e.g., 1% BSA in PBS). A higher blocking volume (200 µl) compared to the reaction volume (100 µl) ensures comprehensive coverage.
• Incubate for 1-2 hours at room temperature or overnight at 4°C.
• Wash with PBS 3-5 times to remove unbound blocking agent.

3. Sample and Standard Addition:

• Add 100 µL of diluted sample (plasma, serum, cell lysates, etc.) to each well.
• Dilute samples appropriately in dilution or blocking buffer to concentrations within the assay's dynamic range (typically 1-100 ng/mL).
• Include positive and negative controls.
• Incubate for 90 minutes at 37°C or overnight at 4°C.

4. Washing:

• Wash the plate several times with PBS to remove any unbound substances.

5. Detection Antibody Addition:

• Add 100 µL of diluted conjugated detection antibody (typically 0.1-1 µg/mL) to each well.
• Incubate for 2 hours at room temperature.
• Wash the plate 3-5 times with PBS to remove unbound detection antibodies.

6. Substrate Addition:

• Add enzyme substrate (e.g., TMB for HRP) to each well.
• Incubate for 15-30 minutes.
• Stop the reaction by adding stop solution (e.g., 1N sulfuric acid for TMB).

7. Reading the Results:

• Measure the optical density of each well using a microplate reader at the appropriate wavelength (e.g., 450 nm for TMB).
• Quantify the antigen concentration by comparing to a standard curve using known antigen standards.

Additional Tips:

• Bring all reagents to room temperature before use.
• Use proper pipetting technique and avoid cross-contamination.
• Adhere to the protocol timings (variations no more than +/- 5 minutes per hour of incubation).
• Avoid repeated freeze-thaw cycles of samples.
• Be consistent with methods used for sample collection and processing.