ELISA Substrates

A Guide to ELISA Substrates

The signal generation step of immunoassay systems is critical to obtaining an accurate measurement of the target protein. Surmodics’ portfolio of BioFX colorimetric and chemiluminescent substrates offers the stability, low background and sensitivity needed to meet the demands of assay manufacturers. A range of substrate sensitivities are available for ELISA, Western Blot, ELISpot and immunohistochemistry applications.

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TMB Substrates

Best-in-class sensitivity, stability and low background in a one-component format for TMB substrates used in manufacturing of immunoassays.

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Explore Surmodics IVD’s ELISA Substrates

ELISA’s (enzyme-linked immunosorbent assay) have been an incredibly useful method in detecting disease for several years. For decades, this plate-based assay has played a major role in identifying and quantifying disease. These protein-based assays use a variety of immunoassay components. Surmodics IVD provides assay developers a one-stop-shop for immunoassay development, including ELISA’s. Learn more about how immunoassay tests work.

Our extensive product portfolio includes protein stabilizers, blockers, diluents, antigens and antibodies, protein stability assay, tmb peroxidase substrate, stop solutions, substrates and more. In the following section we will take a deeper look at our line of ELISA Substrates specifically.

Detection limit, dynamic range, and reproducibility are cornerstones in the development of a successful immunoassay application. During the optimization process, antibody/antigen systems are carefully chosen to provide the specificity and sensitivity of the desired analyte measurement. However, the choice of enzyme/substrate can also have a substantial effect on achieving the above-mentioned parameters and require equal attention for optimal selection.

Criteria for Choosing ELISA Substrates:

During assay development it is important to consider your substrate selection carefully and to have an understanding of your assay’s requirements. Several key elements can be important such as dynamic range, kinetic rate, and assay timing.

Dynamic range is especially important when developing an assay that requires quantitation of a large range of analyte concentrations. Kinetic rate is also an important criterion when finding the right substrate for your ELISA. As such, Surmodics IVD offers several TMB substrates at varying kinetic rates to ensure we have an option suitable for each unique assay in question.

In addition, the timing of your assay is important when considering factors such as dynamic range, reproducibility and detection limit. Assay timing can be optimized by choosing a TMB substrate with the kinetic rate that allows your assay to be performed within the desired timeframe.

Key assay parameters, such as dynamic range, kinetic rate and assay timing are important factors to consider when evaluating various TMB substrates. Each TMB substrate impacts these parameters differently. Choosing the best substrate for your assay will allow the assay developer to create a robust assay with superior performance.

For more information on our recommendations for ELISA substrates, visit the link below.

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TMB ELISA Substrates:

TMB (tetramethylbenzidine) is a chromogenic substrate that is common in numerous staining procedures used at the Horseradish Peroxidase (HRP) detection step. One of its most common applications is in the ELISA procedure. Surmodics IVD’s TMB ELISA Substrates are all offered in ready-to-use formulations that incorporate easily into the existing assay manufacturing process. The Surmodics IVD TMB microwell substrate family outperform competitors for low background, stability and sensitivity with a variety of kinetic rate substrates. Our one-component formulations provide optimal signal generation, longer shelf lives and the ability to streamline manufacturing of assays.

Furthermore, there are several technical advantages when using Surmodics IVD’s TMB ELISA Substrates that can be see in their stability, sensitivity and their lot-to-lot consistency. When looking at sensitivity, Surmodics IVD’s TMB ELISA substrates provide up to 4 years of shelf life stability and prolonged room temperature stability.

In addition, our TMB ELISA substrates also offer assay developers increased sensitivity. Furthermore, they offer increased signal-to-noise ratios and the ability to quantitate through the desired assay range. Our TMB ELISA substrates are also offered in multiple formulations with different kinetic rates that satisfy the measure able range requirements of any assay. Along with their stability and sensitivity, our TMB ELISA substrates also help to eliminate backgrounds yielding lower error through any assay system.

Along with all Surmodics IVD’s immunoassay reagents and elisa reagents, our TMB ELISA substrates are manufactured in our ISO 13485:2016 and 9001: 2015 certified facility allowing us to ensure lot-to-lot consistency and quality.

Learn more about TMB peroxidase substate

Surmodics IVD’s TMB ELISA Substrates:

  • BioFX™ TMB Conductivity One Component HRP Microwell Substrate
    • Product Code: TMBC
    • Mid-kinetic range substrate for ELISA applications.
  • BioFX TMB Super Sensitive One Component HRP Microwell Substrate
    • Product Code: TMBS
    • Gives assay developers maximal signal generation and low backgrounds while maintaining a long shelf life.
  • TMB One Component HRP Microwell Substrate
    • Product Code: TMBW
    • Gives assay developers the best possible tool to maximize shelf life stability and lower backgrounds while setting the industry standard for signal generation of the standard curve.
  • TMB Extended Range HRP Microwell Substrate
    • Product Code: TMBX
    • Low-kinetic range substrate for ELISA applications.
  • TMB Double Slow One Component HRP Microwell Substrate
    • Product Code: TMDS
    • low-kinetic range substrate for ELISA applications.
  • TMB Slow Kinetic One Component HRP Microwell Substrate
    • Product Code: TMSK
    • mid-kinetic range substrate for ELISA applications.
  • TMB Super Slow One Component HRP Microwell Substrate
    • Product Code: TTMB
    • low-kinetic range substrate for ELISA applications.

Visit the link below to learn more about our TMB ELISA substrates and additional TMB substrates.

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Substrate for HRP

Horseradish Peroxidase (HRP) plays an important role in numerous ELISA procedures. TMB is made up of two separate components. One is H2O2, which is hydrogen peroxide and the substrate for HRP. The other component is tetramethylbenzidine (TMB), which generates the blue product.

When the enzymatic reaction takes place, it needs to be stopped at a set point. Usually, this process is completed by adding a strong acid. Many labs use sulfuric acid, H2SO4. Then the substrate turns yellow and it can be measured underneath a spectrometer. This method is popular because it has a high turnover rate, which means that there is a fast color development. It is faster than the alternative, Alkaline Phosphatase (AP).

Substrate for AP

When it comes to Alkaline Phosphatase (AP) the most common substrate is pNPP. pNPP yields a yellow solution that is able to absorb light at 405 nm. As alluded to above, a ready-to-use form of this substrate is the most popular and convenient form.

Surmodics IVD’s AP Yellow One Component Microwell Substrate (pNPP) with Stabilizer Pellets is an excellent option for AP based applications. The formulation consists of p-nitrophenylphosphate (p-NPP) solution with stabilizing pellets to for long term stability. The product is stable for 3 years from the date of manufacture.

Additional ELISA Substrates:

In addition, Surmodics IVD also offers another ready-to-use ELISA substrate. Our ABTS (2,2’-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]) substrate is supplied as a one component, ready-to-use solution for ELISA applications. A soluble blue-green reaction product is obtained when this substrate system is reacted with horseradish peroxidase.

Visit the links below to learn more about Surmodics IVD’s pNPP and ABTS substrates.

Surmodics IVD’s pNPP Substrates Surmodics IVD’s ABTS Substrates