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LI-COR REAGENTS
ELISA Substrates
Reagents for LI-COR Imaging Systems
ELISA Substrates
LI-COR's proprietary peroxidase and phosphatase substrates are optimized for use in the near infrared (NIR) fluorescent region (700 nm) on Odyssey® and Odyssey Sa Infrared Imaging Systems.
NIR fluorescence-based detection also helps overcome the limitation of colorimetric substrate detection, which does not allow for quantification of greater than four optical density units. NIR-based detection allows for a wider linear range.
LI-COR ELISA HRP Substrate (680) and ELISA AP Substrate (680) provide excellent signal-to-noise, consistency, and linearity, making them ideal for use in most ELISA applications. These substrates offer equal or better sensitivity as compared to commercially-available chromogenic and chemiluminescent substrates and are ideal for endpoint assays.
NOTE: These products will not work if used for Western blotting applications.
Figure 1. Substrate Linearity Comparison of TMB to LI-COR ELISA HRP Substrate (680) (P/N 926-34300). Signal-to-noise ratios were determined by dividing the background subtracted average value by the standard deviation of the average background. Limit of detection (LOD) was determined as any SNR value above 3. LOD (0.019 ng/mL) was identical for both substrates.
Figure 2. Serial dilutions of rabbit IgG detected with Goat Anti-Rabbit-HRP followed by LI-COR ELISA HRP Substrate (680) (P/N 926-34300). Row 1 (blank) was coated with PBS only. Rows 2-5 were coated with serial dilutions (5000 - 0.0006 ng/mL) of rabbit IgG in PBS (rows 2 and 3 are duplicates and rows 4 and 5 are duplicates). Imaged on the Odyssey Infrared Imager.
Application Overview
An ELISA (Enzyme-Linked Immunosorbent Assay) is a laboratory method used to detect the presence of an antibody or a protein/antigen in a sample. Also referred to as an EIA (Enzyme Immunoassay), the ELISA is a powerful immunological technique used in many research laboratories as well as industrial labs.
General workflow for the ELISA technique is as follows:
- An unknown amount of antigen is immobilized to a surface (usually a polystyrene microplate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in what is referred to as a "sandwich" ELISA).
- Once the antigen is immobilized, the detection antibody is added. This antibody is conjugated to an enzyme or, alternatively, can be detected by a secondary antibody linked to an enzyme.
- Plates are washed between each step with a mild detergent to remove non-binding antibody.
- A substrate is applied that is converted by the enzyme to a detectable signal, usually a color change.
Products
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ELISA HRP Substrate (680) Pack (P/N 926-34300)
The peroxidase (HRP) substrate results in a colorless to light blue product in the presence of increasing amounts of horseradish peroxidase. The reaction is stopped with a colored peroxidase stop solution, so the wells appear pink after the addition of stop solution.
Pack contains:
- Horseradish Peroxidase (HRP) Substrate (1 vial)
- Assay Buffer (125 mL)
- Stop Solution (25 mL; adequate for 10 x 96-well plates)
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ELISA AP Substrate (680) Pack (P/N 926-34301)
The phosphatase (AP) substrate results in a colorless to light blue product in the presence of increasing amounts of alkaline phosphatase. The reaction is stopped with a colored phosphatase stop solution, so the wells appear purple after the addition of stop solution.
Pack contains:
- Alkaline Phosphatase (AP) Substrate (1 vial)
- Assay Buffer (125 mL)
- Stop Solution (25 mL; adequate for 10 x 96-well plates)
