Description
General Sandwich ELISA Protocol for 96-well ELISA Plates Streptavidin HRP is a useful second-step reagent for labeling a biotinylated primary antibody for detection by ELISA (with an appropriate substrate system). Investigators are encouraged to titrate this reagent, with a suggested starting dilution of 1:1000. Materials needed: Binding Solution Phosphate-buffered saline (PBS) pH 7.2. Note that the pH of the PBS can be increased or decreased or other buffers can be used to obtain better binding of a Capture antibody as experimentally determined. PBS/Tween® Add 0.5 ml of Tween®-20 in 1 L of 1× PBS for a 0.05% solution of Tween®-20. Blocking Buffer: Prepare 10% fetal bovine serum (FBS) or 1% BSA (immunoassay grade) in PBS. The Blocking Buffer should be filtered to remove particulates before use. Blocking Buffer/Tween®: Prepare 10% fetal bovine serum (FBS), 10% newborn calf serum (NBCS) or 1% BSA (immunoassay grade) in PBS/Tween®. The Blocking Buffer should be filtered to remove particulates before use. Chromogenic Substrates Prepare substrate solutions with a commercially available HRP substrate: 1) TMB (3,3',5,5'-Tetramethylbenzidine) is a water-soluble substrate that yields a blue-colored end product upon reaction with HRP. Its color development is faster than ABTS, an alternative HRP substrate. Recommend use of the BD OptEIA™ TMB Substrate Reagent Set (Cat. No. 555214). 2) ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is a water-soluble substrate that yields a green-colored end product upon reaction with HRP. Its color development is slower than TMB, an alternative HRP substrate. Capture antibody: 1. Dilute the purified analyte-specific Capture antibody to ~1-4 μg/ml in Binding Solution. Add 50 μl of diluted antibody to the wells of an enhanced protein-binding ELISA plate. 2. Seal plate to prevent evaporation. Incubate overnight at 4°C. Blocking: 3. Bring the plate to room temperature (RT), wash 3 times with PBS/Tween®, and block nonspecific binding by adding 200 μl of Blocking Buffer per well. 4. Seal plate and incubate at RT for 1-2 hr. 5. Wash ≥ 3 times with PBS/Tween®. Standards and Samples: 6. Add dilution series of the standard analyte and test samples diluted in Blocking Buffer/Tween® at 100 μl per well. 7. Seal the plate and incubate it for 2-4 hr at RT or overnight at 4°C. 8. Wash ≥ 4 times with PBS/Tween®. Detection antibody: 9. Dilute the biotinylated analyte-specific Detection antibody to 0.5-2 μg/ml in Blocking Buffer/Tween®. Add 100 μl of diluted antibody to each well. 10. Seal the plate and incubate it for 1 hr at RT. 11. Wash ≥ 4 times with PBS/Tween®. Streptavidin-Horseradish Peroxidase (Streptavidin-HRP): 12. Dilute the Streptavidin-HRP conjugate (Cat. No. 554066) to its pre-titered optimal concentration in Blocking Buffer/Tween®. Add 100 μl per well. 13. Seal the plate and incubate it at RT for 30 min. 14. Wash ≥ 5 times with PBS/Tween®. Chromogenic Substrate: 15. Dispense 100 μl of TMB using the BD OptEIA™ TMB Substrate Reagent Set (Cat. No. 555214) or ABTS substrate solutions into each well. Incubate at RT (5-45 min) and monitor for color development. The color reaction can be stopped by adding commercially available Stopping Solution at 50 µL per well. 16. Read the optical density (OD) for each well with a microplate reader. The colored product generated from TMB by HRP is read at 450 nm and if wavelength correction is available, 450-570 nm is recommended. The colored product generated from ATBS by HRP is read at 405 nm and if wavelength correction is available, 405-490 nm is recommended. 17. The intensity of the colored product is proportional to the amount of analyte present in the test sample. Using an ELISA reader and ELISA software, plots of absorbance versus a dilution series of standard analyte concentrations are used to calculate analyte levels present in samples. Warning: Streptavidin HRP contains 0.004% (w/w) of a CMIT/MIT mixture (3:1), which is a mixture of: 5-chloro-2-methyl-4-isothiazolin-3-one [EC No 247-500-7] and 2-methyl-4-isothiazolin-3-one [EC No 220-239-6] (3:1). Hazard statements May cause an allergic skin reaction. Precautionary statements Wear protective gloves and eye protection. Wear protective clothing. Avoid breathing mist/vapours/spray. If skin irritation or rash occurs: Get medical advice/attention. IF ON SKIN: Wash with plenty of water. Dispose of contents/container in accordance with local/regional/national/international regulations.
Streptavidin was conjugated with the enzyme under optimal conditions. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
4. Hazardous Ingredient: ProClin™ 150. Avoid exposure to skin and eyes and ingestion. Wash exposed skin with soap and water. Flush eyes with water.
5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
6. ProClin is a trademark of Rohm and Haas Company.
7. For U.S. patents that may apply, see bd.com/patents.
