Enzyme Substrates for ELISA

While optimization is required to get the best results with any assay system, the following guides provide the general information necessary for selecting an appropriate substrate and choosing initial primary and secondary antibody dilutions.

ELISA substrates differ in price, ease-of-use, sensitivity (i.e., lower limit of detection) and compatibility with imaging equipment. More sensitive substrates cost more, so it might be natural to assume that one should use the most sensitive substrate that can be afforded. However, this is not necessarily correct because ease-of-use is an equally important consideration.

ELISA substrates require a variety of procedures for preparing the substrate working solution and detecting the signal. Some substrates are ready to use, while others require additional materials to be supplied. In addition, each substrate has unique requirements for optimization. In general, it is more difficult to optimize ELISA protocols with more sensitive substrates because even the tiniest flaws in blocking or non-specific binding are detectable. No researcher wants to spend more time than absolutely necessary optimizing antibody dilutions and reading times to obtain useful results. Therefore, choose an ELISA substrate that will work at or slightly below the limits of detection required for a specific assay and then optimize.

• Download Tech Tip #33: Guide to Enzyme Substrates for ELISA.

We offer one colorimetric (also called chromogenic) substrate for ELISA development with alkaline phosphatase (AP) and three substrates for horseradish peroxidase enzyme (HRP):

• PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. PNPP produces a yellow water-soluble reaction product that absorbs light at 405 nm. PNPP is available either as a crystalline powder, 5 mg tablets or as a ready-to-use formulation.
• ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is used to detect HRP and yields a water-soluble green end reaction product. The green product has two major absorbance peaks, 410 nm and 650 nm. ABTS is less sensitive than the OPD and TMB substrates for HRP detection. Color development is slow (approximately 20 minutes) which may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities. ABTS is available in either tablet or a ready-to-use for formulation.
• OPD (o-phenylenediamine dihydrochloride) is used to detect HRP and yields a water soluble yellow-orange reaction product. The reaction product has an absorbance maximum of 492 nm. OPD is available in either powder or tablet forms and easily prepared by dissolving in Stable Peroxide Substrate Buffer or buffered hydrogen peroxide solution.
• TMB (3,3',5,5'-tetramethylbenzidine) soluble substrates yield a blue color when detecting HRP. The major absorbance maxima or the reaction product are 370 nm and 652 nm. The color then changes to yellow with the addition of sulfuric or phosphoric acid with maximum absorbance at 450 nm. TMB is very sensitive and may produce significant background signal if too much protein or antibody is used. TMB is more quickly oxidized than other HRP substrates, resulting in faster color development.

Substrates for use with an absorbance plate reader listed in order of increasing sensitivity.

We offer several chemiluminescent substrates for ELISA development with horseradish peroxidase enzyme (HRP):

• CSPD and CDP-Star substrates are chemiluminescent alkaline phosphatase substrates, ready-to-use for protein or nucleic acid blotting on nitrocellulose membranes. These substrates can also be used in solution-based assays. They are supplied at 5 or 25 mM (respectively) in aqueous buffer. See details about improving sensitivity with either Sapphire-II or Emerald-II luminescence enhancers.
  
• DynaLight Substrate with RapidGlow Enhancer is a ready-to-use chemiluminescent substrate formulation that has been optimized to achieve faster results in solution-based assays. The DynaLight Substrate with RapidGlow Enhancer formulation includes 1,2-dioxetane chemiluminescent substrate and a polymeric enhancer that enables ultra-sensitive immunoassay detection by alkaline phosphatase label.
• SuperSignal ELISA Pico Chemiluminescent Substrate provides excellent performance for a large range of target protein amounts and is easily optimized to detect with greater senstivity than entry-level colorimetric substrates. Rapid signal generation with 5-30 minute signal stability depending on HRP concentration.
• SuperSignal ELISA Femto Maximum Sensitivity Substrate is one of the most sensitive substrates available for ELISA applications. When properly optimized, the lower detection limit is 1 to 10 orders of magnitude lower than commonly used colorimetric substrates. However, without proper optimization, it easy to overwhelm the system with protein and enzyme, resulting in high background and possibly negative results.

Substrates for use with a luminometer or other plate reader capable of measuring total luminescence without the use of filters and excitation wavelength listed in order of increasing sensitivity.

We offer two chemifluorescent substrates for ELISA development with horseradish peroxidase enzyme (HRP):

• QuantaBlu Fluorogenic Substrate has a larger linear detection range with low-end linearity for detection of HRP. The stable fluorescent reaction product has an Emax/Amax of 420 nm/325 nm allowing stopped, non-stopped and kinetic assays to be performed; an advantage over the more sensitive chemiluminescent substrates.
• QuantaRed Enhanced Chemifluorescent Substrate is the most sensitive fluorescent ELISA substrate available for HRP detection. The fluorescent reaction product (resorufin) is stable for 4 hours with an Emax/Amax of 585 nm/570 nm when the reaction is stopped. The red-shifted resorufin reaction product permits detection at a wavelength less interference from autofluorescence that can occur in biological samples.
• Amplex Red Reagent a highly sensitive and stable probe for H2O2, is one of our best fluorogenic substrate for peroxidase. Because H2O2 is produced in many different enzymatic reactions, the Amplex Red reagent allows researchers to detect the activity of many different enzymes (Amplex Red Assay Kits).
• Amplex UltraRed Reagent improves upon the performance of our unique Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays.

Substrates for use with a fluorescent plate reader listed in order of increasing sensitivity.