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HRP and other 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 an 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 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.
We offer one colorimetric (also called chromogenic) substrate for ELISA development with alkaline phosphatase (AP) and three substrates for horseradish peroxidase enzyme (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 |
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 |
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 |
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 |
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 |
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 |
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 |
HRP | Novex TMB Chromogen Solution(500ml) Stabilized Chromagen(25ml) Stabilized Chromagen(1L) | 00-2023 SB01 SB02 | 450nm (652nm) Yellow (Blue) | ||
Gal | ONPG Substrate Powder (5g) | 34055 | 410nm Yellow | ~10ng/well (100ng/mL) | 1° 1:500 2° 1:5K |
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. |
We offer several chemiluminescent substrates for ELISA development with horseradish peroxidase enzyme (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 |
---|---|---|---|---|---|
AP | CDP-Star Substrate | ||||
AP | CSPD Substrate | ||||
AP | DynaLight Substrate with RapidGlow Enhancer | 4475406 (100ml) 4475410 (1L) | 1 pg/ml | ||
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 |
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 |
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. |
We offer two chemifluorescent substrates for ELISA development with horseradish peroxidase enzyme (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 |
HRP | QuantaBlu Kinetic Fluorogenic Substrate | 15162 (2700 wells) | 420nm / 325nm | ~500fg/well (5pg/mL) | 1° 1:500 2° 1:5K to 1:20K |
HRP | QuantaRed Enhanced Chemifluorescent Substrate | 15159 (1000 wells) | 585nm / 570nm | ~400fg/well (4pg/mL) | 1° 1:1K 2° 1:5K to 1:20K |
HRP | Amplex Red Reagent | A12222 (200 assays) | 571 / 585nm | ||
HRP | Amplex UltraRed Reagent | A36006 (3400 rxns) | 490–550 nm/580–590nm | ||
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. |
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