Exazym^® Biotin Detection Kit

FAQs

1. We have Thiomersal or sodium azide as preservatives in the antibody solution. Will it have an impact on the conjugation reaction?

   Typically, concentrations of sodium azide (≤ 3 mM or 0.02%) or thimerosal (≤ 0.02 mM or 0.01%) do not significantly interfere with NHS-ester reactions. However, higher concentrations can cause interference. We recommend you remove it prior to conjugation. Removal is performed using one of the spin columns provided in the Exazym® ClickChem Conjugation Kit.

2. We have BSA (bovine serum albumin) in the detector antibody solution. Will it have an impact on the conjugation reaction?

   BSA is typically used at a much higher concentration than the antibody it protects. Because BSA contains lysines, it will compete with the conjugation of the oligo-dT Primer to the detector antibody. For the best results, we recommend to use an antibody solution without BSA or remove BSA using a BSA Removal Kit, such as ab173231 from Abcam.

3. How is the Exazym® oligo-dT Primer conjugated to the detector antibody of the standard immunoassay?

   Conjugation of the Exazym® oligo-dT Primer to the detector antibody in the immunoassay is performed using click chemistry. Specifically, we employ a Cu-free click reaction based on strain-promoted alkyne−azide cycloaddition (SPAAC). The conjugation process involves two steps:

   1. Labeling the Detector Antibody with azide: Initially, the detector antibody is labeled with the ClickChem Label (NHS-PEG4-azide). The ClickChem Label reacts with primary amines on the detector antibody. These primary amines are present at the N-terminus (α-amino group) of each polypeptide chain and in the side-chain of lysine amino acid residues (ε-amino group). Because of their positive charge at physiological pH, they predominantly occur on the external surfaces of native protein tertiary structures, making them readily accessible for conjugation.

   2. Conjugation of the DBCO-Modified Exazym® Oligo-dT Primer: In this step, we add the DBCO-modified Exazym® oligo-dT Primer, which reacts with the azide-labeled detector antibody. As a result, the primer becomes conjugated to the detector antibody.

4. Why are spin columns included in the Exazym® ClickChem Conjugation Kit?

   In the small kit, we provide two spin columns. One of these columns can be used for buffer exchange of the unmodified detector antibody. This is particularly useful if there are substances that interfere with amine coupling, such as buffer ions with primary amines (ex. Tris and Glycin) or preservatives like azide or thimerosal. The other spin column is used to remove free uncoupled/deactivated ClickChem label prior to conjugation of the oligo-dT Primer to the ClickChem labeled detector antibody.

5. Does the PBS-T buffer supplied with the Exazym® ClickChem Conjugation Kit, used to prepare the detector oligo-dT Primer conjugate, contain any added preservatives?

   No preservatives are added to the PBS-T buffer. We have intentionally left it preservative-free to allow users to choose and add their own preservatives if needed.

6. Are there anything in the BOLD components that can disturb the antibody-antigen interactions?

   It is not expected that the BOLD components will disturb antibody-antigen interactions, as antigen binding has already occurred by the time signal amplification with BOLD begins. However, the conjugation of the Exazym® Oligo-dT Primer to the detector antibody of the immunoassay may affect its binding to the antigen. This potential impact should be assessed.

7. Is it possible to prepare the Exazym® RT Polymerase working solution in advance or does it have to be made shortly before use?

   We recommend preparing the working solution shortly before use. However, we have tested storing it overnight in the refrigerator (2-8°C) and found it has very little effect on the assay signal.

8. Is it possible to prepare the Reaction solution with Template in advance or does it have to be made shortly before use?

   We recommend that the mixture of Reaction solution and Template is prepared shortly before use, since we have observed for some assays that the signal decreases if the solution is prepared in advance.

9. According to the IFU Exazym® Antibody Biotin should be incubated 1 hour in the Antibody buffer before use. Does it have to be exactly 1 hour?

   No, it does not have to be exactly 1 hour. However, in our tests, we have observed that pre-incubating the Antibody Biotin in the Antibody Buffer for 30 to 75 minutes reduces the background signal compared to using it immediately after mixing. There is no further improvement beyond 75 minutes. For consistency and reproducibility, we recommend using the same pre-incubation time for all assays.

10. We use PBS-Tween as a wash buffer in our standard immunoassay. Can we use PBS-T as a wash buffer for washing after the BOLD steps?

    Yes, you can use PBS-T, containing 0.05 % Tween™ 20, without any negative effect on BOLD or other steps after BOLD.

11. Can smaller or larger volumes of the BOLD reagents than 100 µL/well be used?

    Yes, other volumes can be used as long as the concentrations of the reagents or reagent mixtures in the volume you will use are as outlined in the Instructions for Use (IFU). For example, if a smaller volume of a reagent mixture is required, the volume of each component should be scaled proportionally to maintain the same concentration, as described in the IFU.

12. Can lids be used instead of adhesive plate covers?

    We recommend using adhesive plate covers as they prevent evaporation and contamination. However, you are welcome to test both options to see which works best for your needs.

13. Can I re-use left-over buffers and reagents in another analytical run?

    We recommend not to re-use mixtures prepared from more than one component/reagent, such as Polymerase working solution, and Reaction Solution and Exazym® Template mix.

14. Is it possible to increase the amplification by increasing the concentration of polymerase?

    The Instructions for Use (IFU) of the Exazym® Polymerase Reaction Kit recommend specific concentrations and incubation times for the polymerase reaction to achieve signal amplification. However, for optimal performance of a BOLD amplified immunoassay, the concentration of polymerase and the polymerization time may need to be adjusted.

15. Is it possible to increase the amplification by increasing the polymerization time to longer than 1 hour?

    This depends on the amplified assay and can be tested. However, if low signal amplification is achieved, it could also be due to other factors within the assay, such as an unsuccessful conjugation of the Exazym® oligo-dT Primer to the detector antibody.

16. Can the incubation steps be done at 37 degrees instead of room temperature?

    For the polymerization step it is not recommended to use 37 degrees since it will lead to a less efficient polymerization with the provided RT polymerase. A custom-made solution with another polymerase reaction kit optimized for 37 degrees is possible. Please contact us if you are interested in such a solution.

17. It looks like something has been sedimented in the Antibody Buffer. Is it ok to use and should I mix it before use?

    Some components of this solution may sediment. Try a few short/quick vortex steps to get a homogenous solution before use.

18. We get a high signal amplification but also a large increase in background. What can we do to reduce the background?

    Try decreasing the concentration of the oligo-dT detector conjugate. Typically, you will need to reduce the concentration of the oligo-dT detector conjugate with some factors compared to the concentration used in the standard immunoassay. Additionally, you can improve the quality of the oligo-dT detector conjugate by removing unconjugated oligo-dT primers. This can be done using an ultra-centrifugal filter unit (e.g., Amicon®) with a suitable cutoff (100 kDa) and volume (0.5 mL). Furthermore, you can evaluate an oligo-dT primer detector conjugate by applying different ratios of the ClickChem label and the oligo-dT primer compared to the detector antibody.

19. We do not have an unmodified detector antibody, we only have access to a biotinylated detector antibody. Can we still use BOLD for signal amplification.

    No, to effectively amplify the signal with BOLD, you need an unmodified detector antibody to which you will conjugate the oligo-dT Primer. Using a biotinylated detector antibody will interfere with the detection of the DNA/RNA hybrid strand. This detection is performed using Exazym® Antibody Biotin (a biotinylated tertiary anti-DNA/RNA antibody) and a streptavidin-enzyme conjugate of your choice, such as streptavidin-HRP.