A Tris-based buffer is recommended for use in DELFIA® assays. Phosphate buffers can be used with N1-chelates, however, the signal may be lower than with Tris-based buffers. For storage purposes, phosphate buffers must not be used due to their chelating nature.
To avoid non-specific binding the buffer should contain a blocking agent such as bovine serum albumin (BSA); using purified BSA is highly recommended. Some grades of BSA contain a considerable amount of heavy metals that will eventually show as high levels of background in the assay. Alternatively, a high grade of casein or ovalbumin may also be used to block the non-specific binding.
A detergent such as Tween 20 or Tween 40 is also needed in the buffer to further prevent non-specific binding to the plate. To keep the fluorescence background low as well as maintain good precision the assay buffer should contain low concentrations of a chelator such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). It is essential to remember that the presence of too much chelator in the assay buffer can compete for the lanthanide chelate and will negatively affect the assay. As a general rule, no more than 50 μmol/L of chelator should be used in the assay buffer when working with compounds labeled with N1-chelate.
An example of an assay buffer composition for a DELFIA assay could be:
50 mM Tris-HCl, pH 7.5-8, containing 0.9% NaCl, 0.2-0.5% of purified BSA, 0.01-0.1% Tween (20 or 40) and 20 μM EDTA.
A variety of different DELFIA buffers and other solutions are offered that are optimized for specific applications or assay steps.
DELFIA Assay Buffer, ready to use
Tris-HCl buffered (pH 7.8) salt solution with 50 mL BSA, bovine globulin, Tween 40, 20 µM DTPA, an inert red dye and <0.1% NaN3 as preservative. Used in DELFIA assays as a binding buffer for lanthanide-labeled antibodies or antigens. It is optimized to give a minimum non-specific background in solid phase assays and is also available without detergents for specific needs, such as cell-based assays.
DELFIA Assay Buffer, without detergents, 5X Concentrate
Same as DELFIA Assay Buffer, supplied as a 5X concentrate without detergents.
DELFIA Wash Concentrate (25X)
25-fold concentration of Tris-HCl buffered (pH 7.8) salt solution with Tween 20. Contains GERMALL® II as preservative. Used to wash DELFIA assay plates prior to enhancement.
DELFIA L*R Binding Buffer Concentrate
500 mmol/L Tris-HCI buffered salt solution (pH 7.5) with 50 mmol/L MgCl2, 250 μmol/L EDTA, 2% bovine serum albumin (BSA) and 0.05% NaN3 as preservative. Optimized to give minimum fluorescence background and non-specific binding in DELFIA ligand receptor binding assays.
DELFIA L*R Wash Solution Concentrate (25X)
250 mmol/L Tris-HCl buffered salt solution (pH 7.5) with 125 mmol/L MgCl2 and 0.05% NaN3 as preservative.
Research Buffer Set
Research Buffer Set is ideal if the assay has some restrictions on the buffer. It contains basic buffer concentrate that can be modified by the user by adding assay specific blocking proteins or detergents – no detergents or proteins (BSA, etc.) are included in the basic buffer concentrate. Bovine serum albumin (BSA) and detergent are included in separate vials.
DELFIA Enhancement Solution
Since DELFIA chelates are not fluorescent, this solution (an acidic chelating detergent solution) is added to the microplate wells after a wash step to develop the fluorescence signal. This provides highly sensitive Eu and/or Sm measurements. Suitable for use in all multianalyte assays.
This Triton X-100, HCl and chelator buffer is an alternative to Enhancement Solution for Eu and/or Sm measurements. For use with stable chelates such as DTPA, to provide more rapid signal development. Suitable for use in all multianalyte assays.
DELFIA Enhancer is used in multianalyte assays using Tb or Dy chelates. After the addition of Enhancement Solution or Inducer, Eu and/or Sm are measured. DELFIA enhancer is then added to the wells and Tb and/or Dy are measured. It is important that Eu and Sm are measured before the addition of DELFIA Enhancer. Eu and Sm form a fluorescent chelate also with DELFIA Enhancer but the intensity of that chelate is not as high as with Enhancement Solution or Inducer.
Stabilizer is BSA which has been further purified to remove heavy metals ions and chelating agents. Its primary function is to stabilize lanthanide-labeled reagents. It may also be used to coat plates, serve as a blocking agent in assay buffer, and act as a component in the storage buffer of lanthanide labeled reagents.
DELFIA labeled reagents must be stored in Tris-HCl buffered solution containing sodium chloride, sodium azide (not necessary when stored at -20°C or -70°C) and BSA (for stabilization). The use of a stabilizer is very important as impure BSA must not be used in the storage buffer of lanthanide-labeled reagents. When stored at a reasonable concentration (e.g. antibodies no less than 10 µg/mL) in this buffer DELFIA labeled reagents are stable for up to 1-2 years. Labeled proteins and peptides should be stored at a high concentration and in the absence of chelators or competing metals in the buffer. It is not recommended to store diluted reagents. In most cases, 50 mmol/L Tris-HCl buffered saline solution (pH 7.5-8.0) containing 0.1-0.5% purified BSA will ensure the stability of the labeled compound during storage. If the labeled protein requires storage at +4°C, it is advisable to add a bacteriostatic agent such as sodium azide (NaN3) at concentration of 0.05-0.1%.
Neither DELFIA Assay Buffer (Prod. No. 1244-106, 1244-111, 4002-0010) nor phosphate buffers are suitable for storage of
labeled proteins or peptides.
We recommend our DELFIA wash concentrate for biochemical DELFIA ELISA assays. If you will be making your own wash buffer, we recommend using a low concentration wash buffer (for example, 10 mM Tris-HCl pH 7.8 with NaCl and Tween) to avoid issues with pH when adding Enhancement Solution after your final wash. Residual wash buffer in the well can potentially alter the pH of the Enhancement Solution at high concentration. Enhancement Solution has pH 3.1 - 3.2. This low pH facilitates the fast dissociation of Europium from its chelate. The Europium then forms a new, fluorescent chelate in Enhancement solution. If the pH of Enhancement solution becomes higher than 3.1 - 3.2 (e.g. pH 4-5), signal enhancement is much slower (but will eventually reach the same signal level as at pH 3.1 - 3.2).
Other buffer tips and FAQs
DELFIA buffers for adherent cells
The critical components in a DELFIA assay with adherent live cells include incubation buffer with Eu-labeled reagent and washing after incubation with Eu-labeled reagent. Assay buffer for Eu-labeled reagent should be Tris-HCl (50 mM) buffered (or Hepes buffered) solution containing 0.9% NaCl (and possibly some other salts), 0.1-0.5% BSA, and 20 µM (micromolar) EDTA or DTPA. Suitable concentration for Eu-labeled antibody is 100-300 ng/mL during incubation. Suitable wash solution is 10-20 mM Tris-HCl pH 7.5 containing 0.9% NaCl. It is necessary for washing to be performed gently and this process should include 4-8 wash cycles. If washing is performed manually then each wash should be 300-350 µL/well.
If you are performing a ligand-binding assay on whole, unfixed adherent cells, the L*R wash concentrate is not recommended because there is no physiological concentration of sodium chloride. For a ligand binding assay using adherent, unfixed cells, we recommend using 10-15 mM Tris-HCl, 0.9% NaCl, pH 7.8 as a wash buffer. No detergent should be present in this wash buffer. You can prepare a 5X concentrate if desired. This wash solution shouldn’t be stored for more than a few days because there is no bacteriostatic agent. If a concentrate is prepared and filtered through 0.45 µm or 0.22 µm membrane, then it should be stable at +4°C at least for a few weeks. For a binding buffer (assay buffer) for use on whole, unfixed adherent cells, we suggest DELFIA Assay Buffer without detergents, 5x concentrate (Cat. No. CR85-100). This assay buffer is Tris-HCl buffered (pH 7.8) salt solution with DTPA, BSA and bovine gamma globulin.
Protocol for fixing cells to a plate
Q: What is the difference between Enhancement solution, Inducer, and Enhancer?
A: DELFIA Enhancement solution is optimal for any N1 chelate (regardless of the lanthanide used). It dissociates the ions quickly and allows fluorescence development for Eu and Sm simultaneously. When a more-stable labeling chelate is needed, we recommend using DTPA chelates for labeling. Inducer provides a more rapid dissociation tool for DTPA labels. DTPA labeled DELFIA reagents could also be enhanced with Enhancement solution, though this would require a longer dissociation time (30 minutes with shaking).
When either Eu or Sm is used as the label, the signal can be measured directly from Enhancement Solution, or Inducer. When Tb is used as an additional label, it is dissociated with Enhancement Solution or Inducer as described above, but also requires the addition of Enhancer, to create a highly fluorescent Tb chelate.
- DELFIA Enhancement solution is for Europium and Samarium N1 chelate dissociation and enhancement, as well as for Terbium N1 chelate dissociation
- DELFIA Inducer is a more-strongly dissociating solution recommended for Europium and Samarium DTPA chelates, as well as for Terbium DTPA chelate dissociation
- DELFIA Enhancer is for Terbium enhancement, after dissociation has been first accomplished with Enhancement solution or Inducer