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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
Matching capture and detector antibodies against human interleukin-4, and with an oligo-dT primer conjugated to the detector antibody. The antibodies can also be used for IL-4 from non-human primates like rhesus macaque and cynomolgus macaque. Exazym® Antibody pair human IL-4 is designed to be used together with Exazym® Polymerase Reaction Kit 96 or 960 and any of the Exazym® Detection Kits.
Components
- 1 vial 80 µg Exazym® IL-4 Capture antibody (clone IL4-I), 1 mg/mL, in phosphate buffer with 0.02% sodium azide
- 1 vial 30 µg Exazym® IL-4 Detector antibody (clone IL4-II), lot specific concentration, in phosphate buffer with 0.02% sodium azide
Shipping conditions
Shipped on wet ice.
Storage conditions
Stored at +2°C to +8 °C.
Performance
Standard range: 0.08 – 20 pg/mL
Documents
Instruction for use
IFU (Article # 10-3011-01)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
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The Exazym® Antibody Pair Human IL-4 Kit contains an IL-4 capture antibody and an IL-4 detector antibody conjugated to an oligo-dT primer. What standard do you recommend for calibration?
The kit does not include an IL-4 standard, and unfortunately, we cannot recommend a specific product to purchase. However, we advise using an IL-4 standard that has been calibrated against the international standard provided by the National Institute for Biological Standards and Control (NIBSC). This ensures consistency and reliability in your quantification.
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What concentrations do you recommend for coating the IL-4 capture antibody and for the IL-4 detector antibody in a BOLD-amplified ELISA?
When using a MaxiSorp™ 96-well plate, we recommend coating with the IL-4 capture antibody at a concentration of 0.25–0.5 µg/mL in phosphate-buffered saline (PBS), pH 7.4. After the coating step, the wells should be washed with PBS + 0.05% Tween-20, followed by a blocking step. We recommend using PBS + 1% casein as the blocking buffer (e.g., Thermo Scientific™ Blocker® Casein, Art. No. 37528). The coating is performed overnight at 2–8°C, followed by a wash step and a 1-hour blocking incubation at room temperature and a final wash step before adding sample or calibrators.
For the IL-4 detector antibody, we recommend a working concentration between 0.025-0.1 µg/mL and a 1-hour incubation at room temperature. We recommend the detector antibody to be diluted in PBS + 0.05% Tween-20 (PBS-T) + 0.1% casein, which corresponds to a 10-fold dilution of Blocker® Casein in PBS-T. This buffer can also be used to dilute calibrators and samples.
The concentrations and buffer compositions provided are suggested starting points. Depending on your specific assay conditions, alternative blocking agents, such as bovine serum albumin (BSA) or non-fat dried milk (NFDM), may offer improved performance and are worth evaluating.
Note: When working with plasma or serum samples, it may be necessary to use an ELISA diluent that blocks heterophilic antibodies to prevent false-positive signals. We have tested Mabtech ELISA Diluent (Art. No. 3652-D2), but further evaluation is needed before we can make a specific recommendation.
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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