Exazym® Polymerase Reaction Kit
Price range: kr 5 050,00 through kr 33 490,00
Reagents intended for performing polymerase reactions when applying BOLD technology for signal amplification of immunoassays.
Pay with credit card or purchase order
First-Time Buyer?
As a first-time user of Exazym®, you will need all three kits. Please select a size above and click below to have all three kits added to your cart.Description
Product Details
Product Specification
Reagents intended for performing polymerase reactions when applying BOLD technology for signal amplification of immunoassays. For research use only. Not for use in diagnostic procedures.
Components
- 1 vial 10 units 20 µl Exazym® Polymerase
- 1 vial 10 ml Exazym® Polymerase Buffer
- 1 vial 95 µl of poly-rA template
- 1 vial 4 ml Exazym® Reaction Solution
Shipping conditions
Shipped on wet ice.
Storage conditions
All components are stored at -20°C.
Shelf life
18 months from date of manufacture. Please refer to the secondary kit packing label for the manufacturing date.
Reagents intended for performing polymerase reactions when applying BOLD technology for signal amplification of immunoassays. For research use only. Not for use in diagnostic procedures.
Components
- 1 vial 100 units 20 µl Exazym® Polymerase
- 1 vial 100 ml Exazym® Polymerase Buffer
- 1 vial 900 µl of poly-rA template
- 1 vial 40 ml Exazym® Reaction Solution
Shipping conditions
Shipped on wet ice.
Storage conditions
All components are stored at -20°C.
Shelf life
18 months from date of manufacture. Please refer to the secondary kit packing label for the manufacturing date.
Documents
Instruction for use
IFU (Article # 10-1001-02) IFU (Article # 10-1001-01)Material Safety Data Sheet
Polymerase Buffer (EU) (Article # 20-1003-01) Polymerase Buffer (USA) (Article # 20-1003-01) Reaction-Solution (EU) (Article # 20-1002-xx) Reaction-Solution (USA) (Article # 20-1002-xx)Certificate of Analysis
Enter your lot number
Application Notes
Development of an Ultra-Sensitive Anti-TNF-a ELISA Using BOLD Technology BOLD Signal Amplification of a Human pTau(181) Sandwich ELISA Signal Amplification of a Human IL-4 Sandwich ELISA Efficient Oligo-dT Primer Conjugation for BOLD Amplified ELISAFAQs about this product
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.
FAQs for all kits
-
We plan to use a streptavidin–horseradish peroxidase (SA-HRP) conjugate and TMB (3,3′,5,5′-tetramethylbenzidine) as substrate for colorimetric detection at 450 nm. Which streptavidin–horseradish peroxidase (SA-HRP) conjugate do you recommend to use?
We’ve had positive experiences using SA-HRP, Art. No. DY998 from Bio-Techne (R&D Systems). When using this SA-HRP conjugate, we recommend a 200-fold dilution, however, depending on the specific system, a higher concentration may be required. As diluent we use phosphate-buffered saline (PBS) supplemented with 0.05% Tween-20 and 0.1% casein (i.e. PBS-T + 0.1% casein).
We incubate the SA-HRP with the bound Exazym® biotinylated antibody for 30 minutes at room temperature, followed by a final wash with PBS-T before adding the TMB substrate. The TMB substrate is then incubated with HRP for 30 minutes at room temperature, and the reaction is stopped with 2 M H₂SO₄. Finally, the absorbance is measured at 450 nm.
-
We’re setting up a BOLD-amplified ELISA as a new assay in a 96-well plate format. Which buffers do you recommend for coating the capture antibody, diluting the sample, diluting the oligo-dT Primer detector conjugate, and for washing?
The following buffer recommendations serve as a starting point and may require optimization depending on your specific ELISA system. In some cases, alternative blockers such as bovine serum albumin (BSA) or non-fat dried milk (NFDM) may provide better performance and are worth evaluating.
- Coating buffer: Phosphate-buffered saline (PBS), pH 7.4
- Wash buffer: PBS + 0.05% Tween-20 (PBS-T)
- Blocking buffer: PBS + 1% casein (e.g., Thermo Scientific™ Blocker® Casein, Art. No. 37528)
- Sample/antigen dilution buffer: PBS-T + 0.1% casein (10-fold dilution of Blocker® Casein with PBS-T).
- Detector conjugate dilution buffer: PBS-T + 0.1% casein (10-fold dilution of Blocker® Casein with PBS-T).
When working with plasma or serum samples, it may be necessary to prepare an ELISA sample diluent that helps prevent false-positive signals caused by heterophilic antibodies. Unfortunately, we are unable to recommend a specific compound or additive to block these antibodies, as effectiveness can vary depending on the sample and assay conditions.
-
We’re setting up a BOLD-amplified ELISA as a new assay in a 96-well plate format. Which plate do you recommend?
We’ve had good experience with Thermo Scientific™ Nunc™ MaxiSorp™ plates or strips with C-bottom wells. These plates are hydrophilic and well-suited for antibody sandwich assays. They are available in clear (for colorimetric detection), white (for fluorescence or luminescence), and black (for fluorescence) 96-well formats, allowing flexibility depending on your detection method.
-
We’ve prepared an oligo-dT Primer detector conjugate and plan to run a BOLD-amplified ELISA. Should we use the same detector concentration as in a standard ELISA?
Not necessarily. While you can start with the same concentration used in your standard ELISA, the signal amplification provided by BOLD may require you to decrease the detector conjugate concentration. We recommend evaluating a lower concentration of the oligo-dT Primer detector conjugate, 2x to 5x is a good starting point to test but since the optimal concentration can vary a lot depending on the antibody/ELISA we recommend testing at least two different concentrations of the detector conjugate.
-
We plan to implement BOLD into a standard ELISA. Do we need to modify our coating, and blocking conditions, or how we add samples?
Typically, no. You can follow your standard protocol for coating, blocking, and sample addition. However, depending on the level of signal amplification achieved with BOLD, you may need to adjust the concentration range of your calibration curve to ensure accurate quantification.
Free consultation for kit providers
Contact us for a no-obligation technical expert consultation to discuss how Exazym® could be integrated into your immunoassay for signal enhancement.
Let's talk