Exazym® StreptaClick® Primer
kr 2 500,00
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Product Details
Product Specification
The product contains the streptavidin-oligo-dT primer required for polymerization of the DNA:RNA hybrid-ladder using the Exazym® Polymerase Reaction Kit. The reagent supplied is sufficient for at least one 96-well microplate. The Exazym® system can bring ultra-sensitive detection levels to conventional immunodiagnostic assays.
Components
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1 vial x 2.5 µg Exazym® StreptaClick® Primer, 0.1 mg/mL in TE buffer.
Shipping conditions
Shipped on wet ice.
Materials and equipment required but not included
- Exazym® Polymerase Reaction Kit 96 or 960
- Exazym® Detection Kit 96 or 960 (select the detection moiety appropriate for your assay; see options above)
- Exazym® Wash Buffer, which is provided as blister tablets with Exazym® Detection Kit 96 and 960.
- ELISA diluent for dilution of Exazym® StreptaClick® Primer (e.g., PBS-based buffer at neutral pH containing stabilizer and detergent)
- Adhesive plate covers
- Plate washer; automated or manual (e.g., multi-pipette)
- Pipettes and other standard laboratory equipment
Storage conditions
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Exazym® StreptaClick® Primer is stored at +2 °C to +8 °C.
Performance
Exazym® StreptaClick® Primer (cat no. 10-0002-01) integrates BOLD signal amplification into any sandwich immunoassay that uses a biotinylated detector antibody. The data below illustrate typical resulting sensitivity gains on two commercial cytokine ELISAs.
Figure 1
Figure 1. Standard curves and limit-of-detection improvement on two commercial assays. Using the optimized conditions, StreptaClick® Primer was applied to the R&D Systems Human IL-23 DuoSet (cat. DY1290, panel A) and the Mabtech Human IL-4 ELISA Flex (cat. 3410-1H-6, panel B). Both standard curves shifted markedly downward on the concentration axis, with LOD (signal-to-noise = 2) improving 11-fold for IL-23 and 16-fold for IL-4.
Figure 2
Figure 2. In-assay optimization and resulting signal-to-noise ratio improvements. Three dilutions of the biotinylated detector antibody were each combined with a high and a low concentration of StreptaClick® Primer; signal-to-noise was evaluated at the manufacturer’s lower specification limit (2 pg/mL) and benchmarked against the unmodified standard ELISA. An 8-fold dilution of detector antibody combined with the higher StreptaClick® Primer concentration delivered the best low-end signal-to-noise ratio.
The degree of sensitivity gain in any assay depends on the extent to which specific signal is amplified as compared to background noise— assays with low intrinsic background see the largest improvements.
Documents
Instruction for use
IFU (Article # 10-0002-01)Material Safety Data Sheet
SAFETY DATA SHEET Exazym® StreptaClick PrimerCertificate of Analysis
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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
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When should I use Exazym® StreptaClick® Primer instead of the Exazym® ClickChem Conjugation Kit?
If your assay uses a biotinylated detector antibody, Exazym® StreptaClick® Primer is a preferred starting point. The Exazym® ClickChem Conjugation Kit is preferred for assays employing detector antibodies which are not biotinylated. Additionally, the Exazym® ClickChem Conjugation Kit provides an opportunity to optimize the detector antibody degree-of-labelling, which can further enhance the performance of Exazym but requires additional development. Finally, the Exazym® ClickChem Conjugation Kit may be preferred when fewer incubation steps are needed or when biotin interference is a concern (e.g., samples with high endogenous biotin).
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Does Exazym® StreptaClick® Primer work with any biotinylated detector antibody, including commercial ELISA kits?
Yes.
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What concentrations should I start with, and how do I optimize?
Two parameters should be cross-titrated for each new assay: the biotinylated detector antibody (recommended 0.125× – 0.5× of the standard protocol) and the StreptaClick® Primer (recommended 10 – 100 ng/mL).
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How much sensitivity improvement should I expect?
Typical improvements range from 10-100X.
FAQs for all kits
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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.
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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.
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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.
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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.
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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.
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