The quantification of a specific marker using an ELISA-based method can be very advantageous when compared to a more qualitative or semi-quantitative method like Western blotting. ELISA is faster, highly sensitive, high-throughput, reproducible and flexible with the ability to analyze a variety of different sample types of diverse origins.
Consistency Low High ELISA can also serve as an orthogonal assay format to other methods such as activity assays or Western blotting to further support and enhance previously reported research results. Drug treatment correlated with the induction of autophagy as indicated by the decrease in p62 levels A and B and by elevation of LC3-II levels C.
The essential components of ELISA are threefold: an antigen to detect and perhaps quantitate, a specific antibody to this antigen, and a system to measure the amount of antigen in a given sample. The crucial component of ELISA-based detection and quantification is the specificity of the interaction between the marker of interest and the antibody. Below, we have highlighted commonly used ELISA assays and their use to determine which is suitable for your research needs.
The surface of the plate is coated directly with the sample. An enzyme-tagged antibody enables its measurement. Incubation is followed by washing, which removes the unbound antibodies from the medium.
The appropriate substrate is then added to the medium, producing a signal directly proportional to the amount of antigen in the sample. This correlation can be used to extrapolate the concentration of antigen in an unknown sample from a standard curve.
Cross-reactivity of the secondary antibody is also eliminated. It is, however, relatively rare as the direct labeling of primary antibodies is time-consuming, expensive, and may adversely affect the immunoreactivity of the antibody with the targeted antigen. In this method, the primary antibody is incubated with the antigen-coated wells.
Next, a labeled secondary antibody that recognizes the primary antibody is added. This secondary antibody is often a polyclonal anti-species antibody. A wide variety of labeled secondary antibodies are readily available. A substrate is then added to the well to produce a signal amplification. This method is commonly utilized to diagnose infection by bacteria, virus, or a parasite and quantify antibodies against this foreign antigen.
Indirect ELISA detection is versatile as different visualization markers can be used with the same primary antibody. However, cross-reactivity and a non-specific signal may occur with the secondary antibody.
Immunometric assays exhibit a direct correlation between antigen concentration and substrate response. In contrast, if there is very little antigen in the sample, there will be very little reduction in the expected signal output. In the example shown in Figure 5, there would be a reduction in signal output. You can create and edit multiple shopping carts Edit mode — allows you to edit or modify an existing requisition prior to submitting.
You will be able to modify only the cart that you have PunchedOut to, and won't have access to any other carts Inspect mode — when you PunchOut to Bio-Rad from a previously created requisition but without initiating an Edit session, you will be in this mode. Third-Party Cookies being blocked. Please Note. Follow us on:. Mainly because all proteins in the sample, including the target protein, will bind to the plate Less flexible - each target protein needs a specific conjugated primary antibody No signal amplification - reduces assay sensitivity Less prone to error — as less reagents and fewer steps are required Best for: when analyzing the immune response to an antigen.
Advantages Disadvantages High sensitivity - more than one labeled secondary antibody can bind the primary antibody Possibility of background noise - secondary antibody may be cross-reactive Longer procedure than direct ELISA technique - additional incubation step for secondary antibody needed Economical - fewer labeled antibodies are needed Greater flexibility - different primary antibodies can be used with a single labeled secondary antibody Best for: determining total antibody concentration in samples.
Advantages Disadvantages High sensitivity - times more sensitive than direct or indirect ELISA Antibody optimization can be difficult - cross-reactivity may occur between the capture and detection antibodies. High specificity - two antibodies are involved in capture and detection Flexibility - both direct and indirect detection can be used Best for: analysis of complex samples, since the antigen does not need to be purified prior to measurement.
Advantages Disadvantages Main advantage - no sample processing is required and crude or impure samples can be used Same limitations as base ELISA - as each ELISA technique can be adapted to a competitive format More robust - less sensitive to sample dilution and sample matrix effects than the sandwich ELISA More consistent - less variability between duplicate samples and assays Maximum flexibility - it can be based on direct, indirect or sandwich ELISA Best for: commonly used when only one antibody is available for the antigen of interest.
It is also suitable for detecting small antigens that cannot be bound by two different antibodies such as in the sandwich ELISA technique. Mainly because all proteins in the sample, including the target protein, will bind to the plate Less flexible - each target protein needs a specific conjugated primary antibody No signal amplification - reduces assay sensitivity.
Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine, and saliva among others. Limited antigen information: information limited to the amount or presence of the antigen in the sample. Each has unique advantages, disadvantages and suitability. Indirect ELISA Indirect ELISA is a technique that uses a two-step process for detection, whereby a primary antibody specific for the antigen binds to the target, and a labeled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection.
No cross-reactivity from secondary antibody. Potential high background: all proteins in the sample bind to the surface. No signal amplification. Low flexibility: the primary antibody must be conjugated. High flexibility: the same secondary antibody may be used for several primary antibodies. Potential cross-reactivity from secondary antibody.
Suitable for complex samples. High flexibility and sensitivity: both direct and indirect methods can be used. Demanding design: finding two antibodies against the same target that recognize different epitopes and work well together can be challenging at times. If an ELISA has been constructed and developed properly, then the intensity of signal produced when the substrate is added will be directly proportional to the amount of antigen captured in the plate and bound by the detection reagents.
Enzyme-conjugated antibodies especially those involving horseradish peroxidase, HRP offer the most flexibility in detection and documentation methods for ELISA because of the variety of substrates available for chromogenic, chemifluorescent, and chemiluminescent imaging.
Colorimetric substrates form a soluble, colored product that accumulates over time relative to the amount of enzyme present in each well. When the desired color intensity is reached, the product absorbance is either measured directly or in some cases a stop solution is added to provide a fixed end point for the assay. Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic ELISA substrates allow direct visualization and enable kinetic studies to be performed.
Furthermore, chromogenic ELISA substrates are detected with standard absorbance plate readers common to many laboratories. The color then changes to yellow with the addition of sulfuric or phosphoric acid, common solutions used to stop the reaction. Chemiluminescence is a chemical reaction that generates energy released in the form of light.
The most common approach is to use luminol in the presence of HRP and a peroxide buffer. The luminol is oxidized and forms an excited state product that emits light as it decays to the ground state. Light emission occurs only during the enzyme-substrate reaction, therefore when the substrate becomes exhausted, the signal ceases. Chemiluminescent detection is generally considered to be more sensitive than colorimetric detection. One drawback of using chemiluminescent substrates for ELISA is that the signal intensity can vary more than with other substrates.
For assays requiring many plates to be read, this can present a problem if the signal begins to decay before plates are read. For this reason, it is important to make sure the assay has been optimized with the substrate in order to avoid misinterpreting signal-fade in a sample as low antigen abundance. Fluorescent ELISA substrates are not as common and require a fluorometer that produces the correct excitation beam to cause signal emission to be generated from the fluorescent tag.
Chemifluorescent detection is also enzyme-based, but the generated product is fluorescent rather than colorimetric. The signal is measured using a fluorometer with the appropriate excitation and emission filters. Chemifluorescence reactions are either measured over time in kinetic assays or halted using a stop solution for direct measurement. In addition to the individual components and general principles of ELISA discussed in this article, ready-to-use ELISA kits are commercially available for detection of hundreds of specific cytokines, chemokines, growth factors, neurobiology analytes, and phosphorylated proteins that are common targets of research interest.
Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. See Navigation. Page contents. ELISA formats direct, sandwich, etc. Direct vs. Plate blocking —addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells. Signal measurement —detection of the signal generated via the direct or secondary tag on the specific antibody. Cross-reactivity of secondary antibody is eliminated. Disadvantages Immunoreactivity of the primary antibody might be adversely affected by labeling with reporter enzymes or tags.
Limited number of conjugated primary antibodies available commercially. No flexibility in choice of primary antibody label from one experiment to another. Minimal signal amplification. Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Different detection methods can be used with the same primary antibody colorimetric, chemiluminescent, etc. Disadvantages Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
An extra incubation step is required in the procedure. Sandwich ELISA Advantages Highly sensitive and highly specific for target antigen as two antibodies are used for capture and detection.
Different detection methods can be used with the same capture antibody.
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