ELISA

Overview

What is an ELISA?

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:

• Immobilization: Provides a means of separating binding partners from unbound molecules in solution. Achieved through adsorption, or adherence to plastic 96-well plate.
• Antigen: The binding partner, or target of the antibody. Typically the immobilized molecule.
• Antibody: At least one polyclonal or monoclonal immunoglobulin which specifically recognizes the antigen.
• Detection: Some means of signaling to quantify bound vs unbound antigen or antibody. Options include colorimetric, fluorescent, or luminescent technologies.

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.

Different ELISA Formats

Direct ELISA	Pro	Cons	Steps	Reagent
	Simple	Requires: High Specificity Ab, Simple Antigen Sample	1. Immobilize	Antigen
			2. Recognize & Detect	Labeled Primary Antibody

Indirect ELISA	Pros	Cons	Steps	Reagent
	High Sensitivity, Modular	Requires: Multi-step, Simple Antigen Sample	1. Immobilize	Antigen
			2. Recognize	Primary Antibody
			3. Detect	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
	Sensitive, Excels with Complex Samples, Reveals Epitopes	Multi-step, Requires Antibody Pair	1. Immobilize	Capture Antibody
			2. Capture Target	Antigen
			3. Recognize & Detect	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
	Reveals Epitopes, Removes Plate-Antigen Interaction, Does Not Require Antibody Pair	Multi-step, Requires Biotinylation	1. Immobilize Avidin	Avidin
			2. Capture Target	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
	Quantifies Antigen in Complex Sample	Multi-step, Requires Standard Curve	1. Immobilize	Antigen
			2. Compete Antigen Recognition Site on Ab	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.

How to Optimize An ELISA

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:

• Plates: Variations in plate chemistry may result in differential antigen adsorption efficiency and non-specific binding, affecting signal to noise ratios. ELISA plate geometries can also affect plating efficiencies and ultimately, final signal strength. Side by side comparison of alternative plate chemistries and geometries should be performed to determine best practices for your ELISA. Should you suspect that plating chemistry is limiting your ELISA performance, testing with a capture ELISA format may prove useful as it removes the potential of epitope masking or conformational changes.
• Buffers: When possible, maintaining optimal temperature, pH, and salt concentrations is critical for optimal performance. This extends beyond binding buffers to coating and wash buffers as well. While higher temperatures may decrease the incubation time, it may also increase non-specific binding. For that reason, room temperature overnight plating, 37 degree Celsius blocking, and return to room temperature for binding may be most appropriate. Again, maintaining all other conditions and testing varying temperatures, pH, salt, irrelevant protein blocking, etc. will reveal the best conditions.
• Antibody Affinity: ELISA performance is highly dependent upon antibody affinity. Different clones will perform better than others at matched concentrations. While affinity is not easily addressed aside from creating or finding alternative antibodies, titrating concentrations will optimize the quantity of antibody used and, within reason, accommodate for even low affinity antibodies. Negative controls are crucial as increasing antibody concentrations risk driving of non-specific binding.
• Antibody Clonality: Use of monoclonal or polyclonal antibodies as capture or detection species will affect the sensitivity of the assay. There are two competing theories regarding antibody clonality in ELISAs. While monoclonal antibodies offer specificity, they typically return lower signal due to the limited epitope recognition. Polyclonal antibodies may recognize more epitopes per antigen, but even with affinity purified polyclonal antibodies they inherently bring with them unwanted cross-reactivity potential. Regarding a sandwich ELISA, while a monoclonal capture antibody will recover only specific targets from a sample, a polyclonal capture antibody will recover the maximal amount of target, but with some non-specific binding. Either clonality of antibody may be used as the detection reagent, again with the same specificity caveats. Sample quality undoubtedly affects ELISA performance. Complex samples would likely benefit from the specificity of monoclonal antibody capture, whereas samples with simple composition, but low target concentration would benefit from a polyclonal antibody capture. Yet again, empirical testing will reveal optimal clonality use, or combinations thereof.
• Signal: Enzymatic signal development provides robust detection, although care should be taken to optimize development time, temperature, and volume prior to quenching the reaction. Too little time or too low temperature may blunt signal generation, whereas too much time or temperature could allow for increased noise. Volumes are normally limited to well sizes, but this too should be maintained constant throughout testing and study periods once optimized. If sufficient signal is detectable, fluorescent reporters may also serve to measure multiple targets simultaneously, with fluorescent labeling of the detection antibody allowing for simplified protocols.

ELISA Applications

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.

ELISA Related Services

Whether generating custom antibodies for an ELISA or bringing ELISA kit production in-house, GenScript is here to help. 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:

• Anti-Idiotype Antibodies making PK or PD testing of therapeutic antibody drugs easy as they only detect the CDR of the target antibody.
• DNA Immunizations, our specialty. Circumvents in vitro protein antigen production and deals with difficult targets.
• High Throughput Gene to Antibody Service providing up to 1 mg of recombinant antibodies in as little as 3 weeks for quick screening.

With 12 years of experience developing customized antibodies, your project is in expert hands. Get a quote today!

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