Preparation And Storage
For extended storage, we recommend the addition of an equal volume of glycerol, making a final concentration of 50% glycerol and reducing the concentration by 50%.
Recommended Assay Procedures
This AKP Streptavidin conjugate can be used to label the biotinylated Detection antibody in ELISA (with an appropriate substrate system). A dilution of 1:1000 is recommended (1:500 if glycerol has been added). For Immunohistochemical staining, we recommend the use of AKP Streptavidin in our special formulation for Immunohistochemistry, BD Pharmingen™ AKP Streptavidin (Cat. No. 551008).
General Sandwich ELISA Protocol for 96-well ELISA Plates
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 the commercially available AKP substrate, P Nitrophenyl Phosphate (PNPP). PNPP is a water-soluble substrate that yields a yellow-colored end product (that absorbs light at 405 nm) upon reaction with AKP. The intensity of the color is proportional to the amount of antibody or antigen present in the test sample, which can be quantified using an ELISA reader and ELISA software.
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 RT, wash 3 times with PBS/Tween®, and block non-specific 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 Alkaline Phosphatase (AKP Streptavidin):
12. Dilute the AKP Streptavidin conjugate (Cat. No. 554065) 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 p-Nitrophenylphosphate (1 mg/ml in 0.05 M K2C03, 1 mM MgCI2, pH 9.8) into each well. Incubate at RT (5-45 min) and monitor for color development.
16. After development of the color reaction, the well contents are measured for absorbance at 410 nm using an ELISA microplate reader. Plots of absorbance measured at 410 nm versus a dilution series of standard analyte concentrations using ELISA software can be employed to calculate analyte levels present in samples.
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.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
Streptavidin is a non-glycosylated protein that is purified chromatographically from the bacterium Streptomyces avidinii. Streptavidin homotetramers have a particularly high, non-covalent binding affinity for biotin. When conjugated with fluorochromes, streptavidin has been widely used with biotin-conjugated primary or secondary antibodies and other biotinylated specific-binding molecules (eg, recombinant proteins and lectins) to stain molecules expressed by cells and tissues for subsequent multiparameter analysis by flow cytometry, fluorescence microscopy and imaging. When conjugated with an enzyme such as Alkaline Phosphatase (AKP) and coupled with the use of a colorimetric, luminescent, or fluorescent substrate development system, AKP Streptavidin has found widespread use along with biotinylated primary or secondary antibodies in a number of applications including Western blot, ELISA, ELISPOT, Immunocytochemistry and Immunohistochemistry.
This reagent conjugate can be used as a detecting antibody in ELISA; a dilution of 1:1000 is recommended (1:500 if glycerol has been added). For immunohistochemical staining, we recommend the use of BD Pharmingen™ AKP Streptavidin (Cat. No. 551008) that is specially formulated for immunohistochemistry. AKP Streptavidin is a useful second-step reagent for labeling a biotinylated primary antibody for detection by ELISA (with an appropriate substrate system).
Development References (3)
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Crowther JR. The ELISA guidebook.. Methods Mol Biol. 2000; 149:III-IV, 1-413. (Methodology: ELISA). View Reference
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Hobbs MV, Weigle WO, Noonan DJ, et al. Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J Immunol. 1993; 150(8):3602-3614. (Methodology: ELISA). View Reference
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Wild, D. Wild, D, ed. The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques. 4th Edition. Waltham, MA: Elsevier Science; 2013:1-1013.
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.