Exazym^® HRP Detection Kit

FAQs for all kits

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.