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ELISA Enzyme Substrates Selection Guide

Choose an appropriate reagent for plate-based assay detection.

ELISA substrate selection guideThermo Scientific Pierce Enzyme Substrates for immunoassays are available in a range of sensitivities for different detection methods. Certain substrates will perform better than others when optimized for a given system and detection limit. For example, a high sensitivity chemluminescent ELISA substrate would be difficult to optimize in a assay where nanogram or microgram amounts of protein are being assayed. In contrast, an entry-level substrate would not provide the sensitivity required to detect femtogram quantities of protein.

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

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Overview of ELISA

ELISA Development

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Assay Development Handbook

Criteria for Choosing ELISA Substrates

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.

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Substrates for ELISA

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Tech Tip #33: Guide to enzyme substrates for ELISA

Thermo Scientific Colorimetric ELISA Substrates

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.

Horseradish peroxidase (HRP) colorimetric ELISA substrates

ABTS Substrates
TMB Substrates
OPD Substrates

Alkaline phosphatase (AP) colorimetric ELISA substrates

PNPP Substrates

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Pierce 96-Well Microplates
Pierce 8-Well Strip Microplates
Reagent Reserviors
Microplate Sealing Tape
Stable Peroxide Substrate Buffer

Colorimetric ELISA Substrates for Alkaline Phosphatase (AP) and Horseradish Peroxidase (HRP). Substrates for use with an absorbance plate reader listed in order of increasing sensitivity.
Enzyme Product Product No.
(Total Assays ¹)
Absorbance
and Color
Detection
Limit ²
Primary (1°) and
Secondary (2°)
Antibody Dilutions ²
AP PNPP Substrate

1-Step Solution
Tablet Kit
Tablets (105)
Powder (25g)


37621 (1000 wells)
37620 (5250 wells)
34047 (5250 wells)
34045 (250,000 wells)
405nm
Yellow
~10ng/well
(100ng/mL)
1° 1:500
2° 1:5K to 1:20K
See Instructions
See Instructions
HRP ABTS Substrate

1-Step Solution
Tablets (50)


37615 (2000 wells)
34026 (3330 wells)
410nm (650nm)
Green
~250pg/well
(2.5ng/mLl)
1° 1:1K
2° 1:5K to 1:50K
See Instructions
See Instructions
HRP OPD Substrate

Powder (25g)
Tablets (50)


34005 (5000 wells)
34006 (5000 wells)
490nm (450nm)
Green (Orange)
~7pg/well
(70pg/mL)
1° 1:1K
2° 1:5K to 1:50K
See Instructions
HRP Slow TMB

1-Step Solution


34024 (2500 wells)
450nm (652nm)
Yellow (Blue)
~8pg/well
(80pg/mL)
1° 1:1K
2° 1:5K to 1:50K
See Instructions
HRP Turbo TMB

1-Step Solution


34022 (2500 wells)
450nm (652nm)
Yellow (Blue)
~7pg/well
(70pg/mL)
1° 1:1K
2° 1:5K to 1:50K
See Instructions
HRP TMB Substrate

2-Part Kit


34021 (4000 wells)
450nm (652nm)
Yellow (Blue)
~6pg/well
(60pg/mL)
1° 1:1K
2° 1:5K to 1:50K
See Instructions
HRP Ultra TMB

1-Step Solution


34028 (2500 wells)
450nm (650nm)
Yellow (Blue)
~2pg/well
(20pg/mL)
1° 1:1K
2° 1:5K to 1:50K
See Instructions
¹ See product instructions for additional information and assay considerations that determine the number assays.
² Detection limits and recommended antibody dilutions (based on 1mg/mL stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.

Thermo Scientific Chemiluminescent ELISA Substrates

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

  • 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.

Chemiluminescent ELISA substrates (for HRP)

SuperSignal ELISA Pico
SuperSignal ELISA Femto

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Overview of ELISA

Chemiluminescent ELISA Substrates for Horseradish Peroxidase (HRP). 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.
Enzyme Product Product No.
(Total Assays ¹)
Emax ³
Color
Detection
Limit ²
Primary (1°) and
Secondary (2°)
Antibody Dilutions ²
HRP SuperSignal
ELISA Pico
37070 (1000 wells)
37069 (2500 wells)
425nm
Blue/Green
~500fg/well
(5pg/mL)
1° 1:1K
2° 1:10K to 1:50K
See Instructions
HRP SuperSignal
ELISA Femto
37075 (1000 wells)
37074 (2500 wells)
425nm
Blue/Green
~170fg/well
(1.7pg/mL)
1° 1:1K
2° 1:50K to 1:100K
See Instructions
¹ Total number of assays based on 96-well microplate. See product instructions for additional information and assay considerations that determine the number assays.
² Detection limits and recommended antibody dilutions (based on 1mg/mL stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.
³ The peak emission wavelength is given for reference. However, for best sensitivity, measure total light output using a luminometer.

Thermo Scientific Fluorescent ELISA Substrates

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

  • QuantaBlu Fluorogenic Substratehas 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 Substrateis 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.

Fluorescent ELISA substrates (for HRP)

QuantaBlu Fluorogenic Substrate

QuantaRed Enhanced Chemifluorescent Substrate

Chemifluorescent ELISA Substrates for Horseradish Peroxidase (HRP). Substrates for use with a fluorescent plate reader listed in order of increasing sensitivity.
Enzyme Product Product No.
(Total Assays ¹)
Emax / Amax
(Emisson/Excitation)
Detection Limit ² Primary (1°) and
Secondary (2°)
Antibody Dilutions ²
HRP QuantaBlu
Fluorogenic
Substrate
15169 (2700 wells) 420nm / 325nm ~500fg/well
(5pg/mL)
1° 1:500
2° 1:5K to 1:20K
See Instructions
HRP QuantaBlu Kinetic Fluorogenic
Substrate
15162 (2700 wells) 420nm / 325nm ~500fg/well
(5pg/mL)
1° 1:500
2° 1:5K to 1:20K
See Instructions
HRP QuantaRed Enhanced Chemifluorescent
Substrate
15159 (1000 wells) 585nm / 570nm ~400fg/well
(4pg/mL)
1° 1:1K
2° 1:5K to 1:20K
See Instructions
¹ See product instructions for additional information and assay considerations that determine the number assays.
² Detection limits and recommended antibody dilutions (based on 1 mg/ml stock) have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.

Coated Plates, ELISA Kits and Other Resources

In addition to the substrate reagents described on this page, many other ELISA reagents (e.g., blocking buffers), pre-coated microplates and complete ELISA kits are available. Coated plates streamline development of many kinds of plate-based assays. Complete kits provide all reagents and optimized protocols for detection of specific cytokines and other targets, such as interferon gamma (IFN gamma) and interleukin 6 (IL-6). ELISPOT kits (enzyme-linked immunospot assay) measure cytokines in single cells.

In-Cell ELISA Kits were developed for performing ELISA assays on plated cells. The Thermo Scientific In-Cell ELISA Kits are available for detection by colorimetric or fluorescent (Near-IR) instrumentation and include all reagents necessary except the primary antibody. The colorimetric version of the In-Cell ELISA is developed using a TMB substrate for HRP, while the Near-IR detection kit uses secondary detection reagents conjugated to DyLight Fluors. Both types of In-Cell ELISA Kits allow multiple target proteins to be compared to each other in the same assay either through normalization or multiplex analysis.

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