- Coomassie Stains & Protein Gel Documentation
- Nucleic Acid Gel Documentation
- ELISA
- EMSA/Gel Shift Assay
- Glycoprotein Detection
- In-Cell Western™ Assay
- In-Gel Western Assay
- Microwestern Array
- Northern Blot
- On-Cell Western Assay
- Protease Assay
- Protein Array
- Quantitative Western Blot
- Reporter Gene Assay
- Reverse Phase (Lysate) Array
- RNAi Analysis
- Small Animal Imaging
- Southern Blot
- Tissue Section Imaging
- Transcription Factor Assay
Email bio-eu@licor.com
APPLICATIONS for the
ODYSSEY® CLx
Applications for the Odyssey CLx Infrared Imaging System
ELISA
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.
LI-COR's proprietary peroxidase and phosphatase substrates are optimized for use in the near infrared (NIR) fluorescent region (680 nm) on Odyssey® CLx, Odyssey Classic, 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.
NOTE: These products will not work if used for Western blotting applications.
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 comparison of LI-COR ELISA HRP Substrate (680) (P/N 926-34300) to various HRP substrates. Substrates were compared in a mouse IgG ELISA for linearity, signal-to-noise ratios (SNR), and limit of detection (LOD). SNR ratios were determined by dividing the background subtracted average value by the standard deviation of the average background. LOD was determined as any SNR value above 3. The LI-COR ELISA HRP Substrate (680) demonstrated superior SNR values and linearity compared to other substrates. The LOD values were 0.313, 0.625, 1.25, and 0.156 ng/mL for ELISA HRP Substrate (680), TMB, ABTS, and LumiGLo.
Figure 2. Substrate Linearity Comparison of Alkaline Phosphatase Substrates to LI-COR ELISA AP Substrate (680) (P/N 926-34301). 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 was 4.9 ng/mL with the ELISA AP Substrate (680) and 9.8 ng/mL with all of the others.
Figure 3. 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.
