# Complete Sandwich ELISA Protocol > Published on Vicena | [View on web](https://vicena.ai/wiki/complete-sandwich-elisa-protocol-ZjTFwA) --- ## User What is the complete sandwich ELISA protocol, including plate coating concentration, blocking buffer choice, primary and secondary antibody dilutions, TMB substrate development, and stop solution? --- ## Vicena A complete sandwich ELISA protocol involves the immobilization of a capture antibody, followed by the sequential addition of the antigen (sample), a detection antibody, and a colorimetric substrate. The following protocol is synthesized from established laboratory standards provided by [Thermo Fisher Scientific](https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/general-elisa-protocol/basic-sandwich-elisa-protocol.html), [Abcam](https://www.abcam.com/en-us/technical-resources/protocols/sandwich-elisa), and [Fortis Life Sciences](https://www.fortislife.com/protocols/elisa-protocols/sandwich-elisa-protocol). ### 1. Plate Coating (Capture Antibody) * **Coating Buffer:** $\ce{0.05 M}$ Carbonate-Bicarbonate ($\ce{pH 9.6}$) or Phosphate Buffered Saline (PBS). * **Antibody Concentration:** $1\text{--}10\ \mu\text{g/mL}$ for affinity-purified antibodies. * **Procedure:** Add $100\ \mu\text{L}$ per well to a high-protein binding microtiter plate. * **Incubation:** 1 hour at room temperature or overnight ($12\text{--}18$ hours) at $4^\circ\text{C}$. ### 2. Blocking * **Blocking Buffer:** PBS or TBS containing $1\text{--}5\%$ Bovine Serum Albumin (BSA) or $5\%$ non-fat dry milk. * **Procedure:** Aspirate coating solution and wash once. Add $200\text{--}300\ \mu\text{L}$ of blocking buffer to each well. * **Incubation:** $1\text{--}2$ hours at room temperature. ### 3. Sample and Standard Addition * **Diluent:** Use the same buffer as the blocking step (e.g., PBS + $1\%$ BSA). * **Procedure:** Wash the plate 3 times with Wash Buffer (PBS + $0.05\%$ Tween-20). Add $100\ \mu\text{L}$ of standards and diluted samples in duplicate. * **Incubation:** $1\text{--}2$ hours at room temperature or overnight at $4^\circ\text{C}$. ### 4. Detection Antibody (Primary/Secondary) * **Primary (Detection) Antibody:** * **Dilution:** Typically $0.1\text{--}2\ \mu\text{g/mL}$ (or $1:1,000$ to $1:10,000$ depending on the datasheet). * **Incubation:** $1\text{--}2$ hours at room temperature. * **Secondary Antibody (if primary is unconjugated):** * **Dilution:** Dilute HRP-conjugated secondary antibody (e.g., $1:2,000$ to $1:10,000$) in blocking buffer. * **Incubation:** 1 hour at room temperature. * **Washing:** Wash 3–5 times with Wash Buffer after each antibody incubation. ### 5. Substrate Development (TMB) * **Substrate:** $3,3',5,5'$-Tetramethylbenzidine (TMB) solution. * **Procedure:** Add $100\ \mu\text{L}$ of TMB substrate to each well. * **Incubation:** $15\text{--}30$ minutes in the dark at room temperature. A blue color will develop. ### 6. Stop Solution and Measurement * **Stop Solution:** $\ce{0.18 M}$ to $\ce{2 N}$ Sulfuric Acid ($\ce{H2SO4}$) or $\ce{1 M}$ Hydrochloric Acid ($\ce{HCl}$). * **Procedure:** Add $100\ \mu\text{L}$ of stop solution directly to the TMB. The color will change from blue to yellow. * **Measurement:** Read the optical density (OD) at $450\ \text{nm}$ using a microplate reader within 30 minutes. ### Summary of Standard Reagents | Parameter | Recommended Specification | | :--- | :--- | | **Coating Conc.** | $1\text{--}10\ \mu\text{g/mL}$ | | **Blocking Buffer** | $1\%\text{--}5\%$ BSA in PBS/TBS | | **Wash Buffer** | PBS + $0.05\%$ Tween-20 | | **TMB Substrate** | Ready-to-use liquid TMB | | **Stop Solution** | $\ce{0.18 M H2SO4}$ | | **Detection Wavelength** | $450\ \text{nm}$ | ---