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"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Janet Duerr, Ohio University
An enzyme linked immunosorbent assay (ELISA) is a robust and sensitive technique used to detect and quantify specific proteins in samples which may contain complex mixtures of proteins. Antibodies are used to detect the specific proteins immobilized on the surface of microplate wells. The technique facilitates high volume and fast throughput analysis, ideal for analyzing large numbers of samples.
ELISAs are performed a number of ways, the most common is the sandwich or capture assay or with labeled streptavidin to amplify analyte signal from a biotinylated antibody.
Common ELISA formats. A. Direct. B. Indirect, C. “Sandwich” capture assay ELISA and D. Streptavidin/ biotin signal amplification using a biotinylated primary antibody.
Blocking reagents are especially important in ELISA, ideally use serum of the species of the labeled antibody to block all unsaturated binding sites on the microplate to avoid background from nonspecific binding, however BSA may be appropriate. See Normal Serums and Bovine serum albumin (BSA) pages. The addition of a mild detergent such as Tween ® 20 can help to reduce background from hydrophobic interactions.
Jackson ImmunoResearch alkaline phosphatase (AP) and horseradish peroxidase (HRP) conjugates can be used for both colorimetric assays, using a chromogenic substrate. For chemiluminescent detection, a luminol based substrate is commonly used with peroxidase conjugates for highly sensitive detection. ELISAs can be performed using fluorescent conjugates to allow simultaneous detection of multiple antigens using primary antibodies derived from different species. Using labeled secondary antibodies each antigen can be distinguished specifically by the individual fluorescent signal. The detection limit for fluorescent ELISA is however typically lower than colorimetric or chemiluminescent detection using a reporter enzyme.
Signal enhancement can be achieved using a labeled streptavidin to detect a biotinylated antibody (primary or secondary antibody for enhanced amplification). Each antibody can itself present multiple biotin molecules, which are then, in turn, able to bind with multiple streptavidin molecules. These combined factors mean that multiple probe molecules are available to either catalyze the detection substrate to its end product or generate fluorescent emission - achieving a brighter signal and greater sensitivity.
Recommended dilution ranges for ELISA are shown in the table below.
|Horseradish Peroxidase Conjugates [IgG, F(ab') 2]||1:5,000 - 1:100,000|
|Alkaline Phosphatase Conjugates [IgG, F(ab') 2]||1:5,000 - 1:50,000|
|Biotin-SP Conjugates [IgG, F(ab') 2] (using enzyme-Conjugated Streptavidin)||1:20,000 - 1:400,000|
|Horseradish Peroxidase-Conjugated Streptavidin||1-2 µg / ml|
|Alkaline Phosphatase-Conjugated Streptavidin||1-2 µg / ml|