ELISA is an acronym for enzyme-linked immunosorbent assay (ELISA). The ELISA is utilized in research, healthcare, and food safety environments to measure target analytes such as hormones, antibodies, and protein biomarkers. In the context of an ELISA analytes are antigens, the targets of antibodies. Antigens are typically adsorbed (attached) to a 96-well plate and specifically recognized by an immunoglobulin (antibody) that has an enzyme linked to it for detection purposes. There are many variations on the ELISA, but the principle requirements are constant:
These fundamental elements can be combined to create many formats including Direct ELISA, Sandwich ELISA, and Capture ELISA. The distinguishing characteristics between them is which component is immobilized, how it is recognized, and what is detected. More elaborate assays can be created through combinations of the 5 basic formats, however for the sake of simplicity, they are outlined below in their most basic form.
Direct ELISA | Pro | Cons | Steps | Reagent |
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Simple | Requires: High Specificity Ab, Simple Antigen Sample |
1. Immobilize
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Antigen |
2. Recognize & Detect
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Labeled Primary Antibody |
Indirect ELISA | Pros | Cons | Steps | Reagent |
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High Sensitivity, Modular | Requires: Multi-step, Simple Antigen Sample |
1. Immobilize
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Antigen |
2. Recognize
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Primary Antibody | |||
3. Detect
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Labeled Secondary Antibody |
Direct ELISA has the simplest format, requiring antigen to be adsorbed to the plate and then bound by a labeled "detection" antibody. "Direct" refers to the first and only antibody acting as both the antigen recognition molecule and signal delivery molecule. This contrasts with an "indirect" ELISA which separates the detection and signaling tasks between "primary" and "secondary" antibodies. An indirect ELISA detects the plated antigen with an unlabeled antibody, which is then detected with a secondary antibody carrying the means of signaling. Whereas the benefit of a direct ELISA lies in its simplicity and speed, the additional steps between antigen binding and signal detection with indirect ELISA formats have their own merits. By utilizing a secondary antibody for signal delivery, indirect ELISA formats allow for use of a modular secondary antibody that recognizes the constant (Fc) region of the primary antibody. Labeled secondary antibodies can therefore be utilized across many different ELISAs and the primary antibody need not be modified. Primary antibodies are typically monoclonal, a precious and expensive resource, whereas secondary antibodies are typically polyclonal, cheaply and quickly produced. Beyond cost, this combination of monoclonal primary and polyclonal secondary also allows for improved performance via signal amplification. As polyclonal antibodies are comprised of many different clones, each recognizing their own epitope, they can bind multiple sites on the Fc region of the primary antibody. In the above example, multiple labeled secondary polyclonal antibodies would decorate the blue primary antibody. A direct ELISA's detection is limited by the degree of labeling of the sole antibody in the assay. An indirect ELISA may bind 2 to 3 secondary antibodies when a polyclonal secondary is used, and therefore possess 2 to 3 fold greater signal per each primary antibody. Direct and indirect ELISA methods may be extended to all the variations discussed below.
Sandwich ELISA | Pros | Cons | Steps | Reagent |
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Sensitive, Excels with Complex Samples, Reveals Epitopes | Multi-step, Requires Antibody Pair |
1. Immobilize
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Capture Antibody |
2. Capture Target
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Antigen | |||
3. Recognize & Detect
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Labeled Detection Antibody |
The differentiating feature of a sandwich ELISA is the adsorption of a "capture" antibody to the plate. Antigen is bound, or captured by the plated antibody and then "sandwiched" between the capture and a detecting antibody which recognizes a distinctly different epitope on the antigen. A major benefit of a sandwich ELISA is the ability to specifically measure antigen from impure samples. Rather than adsorb a crude sample to the plate, capture antibodies provide the assay specificity and decontamination qualities. The opportunity for indirect detection is also available in a sandwich ELISA. The detection antibody would not carry the signal but rather be targeted by yet a third antibody which would impart the signal to the assay.
Capture ELISA | Pros | Cons | Steps | Reagent |
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Reveals Epitopes, Removes Plate-Antigen Interaction, Does Not Require Antibody Pair | Multi-step, Requires Biotinylation |
1. Immobilize Avidin
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Avidin |
2. Capture Target
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Biotinylated Target | |||
3. Recognize & Detect
|
Labeled Detection Antibody |
A similar technique to sandwich ELISA is a "capture" ELISA which uses an avidin biotin complex to retain antigen to the plate. Adsorption to ELISA plates requires some degree of hydrophobic and charge interactions which may negatively affect the structure of the antigen, and subsequently inhibit antibody recognition. By adsorbing large tetrameric protein avidin, biotin labeled antigen can be immobilized , yet avoid adverse plate-antigen interaction. Additionally, avidin-biotin-capture distances the antigen from the plate. Increasing the distance from the plate allows for 3 dimensional access to the antigen whereas direct plating could sterically obscure access to epitopes. The avidin capture method could also be an advantageous technique for recovering biotinylated antigens from complex samples.
Competitive ELISA | Pros | Cons | Steps | Reagent |
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Quantifies Antigen in Complex Sample | Multi-step, Requires Standard Curve |
1. Immobilize
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Antigen |
2. Compete Antigen Recognition Site on Ab
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Antigen and Labeled Detection Antibody | |||
3. Recognize & Detect
|
Labeled Detection Antibody |
The final possible ELISA category is "competitive". It is similar to a direct ELISA in that antigen can be directly adsorbed to the plate. However, the detecting antibodies are pre-incubated with samples containing unknown amounts of antigen prior to application to the plate. Samples with more antigen will occupy binding sites on the primary antibody, thereby blocking it from binding to plated antigen. Conversely, samples with less antigen will have more antibodies available to bind to the plated antigen and return a higher signal. In a competitive ELISA, the signal returned is inversely related to the concentration of antigen-antibody interaction in the sample. Titration of known amounts of antigen to create a standard curve on the same plate is required to quantify available antigen in the unknown samples. The competitive technique can be merged with both capture and sandwich formats. Indirect detection can also be utilized.
Next to western blotting, the ELISA is one of the most common and sensitive molecular biology tools available. Limits of detection in a properly optimized ELISA regularly reach femtogram quantities of antigen. Contrary to Western blots, ELISAs provide highly quantifiable data through comparison of experimental samples to standard curves created by titrating known amounts of sample in matched buffer compositions (eg. including serum if experimental sample is in serum). Beyond choosing the appropriate format, each of the many steps in an ELISA is an opportunity for optimization. Empirical testing of some or all of the following may be required to gain maximum signal-to-noise ratio:
Application Field | Measures |
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Research |
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Healthcare |
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Pharmaceutical Industry |
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Food Industry |
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Drug Abuse Screening |
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Beyond the above list of applications, ELISA technology is even found in common, over-the-counter diagnostics such as home pregnancy tests. These types of tests are termed "dip-stick" ELISAs which utilize lateral flow and the principles of a sandwich ELISA. Capillary action first draws sample through a region which contains unbound detection antibody and then through a region which has immobilized capture antibodies also specific for the analyte. While this simplified version of the ELISA does not offer quantifiable results, the high speed and low cost makes it ideal for use in point-of-care and home testing environments.
GenScript provides customized antibody solutions specifically for ELISA, supporting your unique assay needs. Our MonoExpress™ service is ideal for clean and clear direct ELISAs while PolyExpress™ service is perfect for generating detection antibodies against your target. Our antibody labeling service seamlessly integrates with any project, making custom ELISA creation easy. We even generate optimized paired antibodies for a custom sandwich ELISA. Some other related services include:
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