ELISA- Enzyme Linked Immuno Sorbant Assay
In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interactionELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins.The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).
TYPES
Initially in a direct ELISA test which is considered to be the simplest type of ELISA the antigen is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block all the other binding sites. While an enzyme is linked to an antibody in a separate reaction, the enzyme-antibody complex is applied to adsorb to the antigen. After excess enzyme-antibody complex is washed off, enzyme-antibody bound to antigen is left. By adding in the enzyme's substrate, the enzyme is detected illustrating the siDirect ELISA, when compared to other forms of ELISA testing, is performed faster because only one antibody is being used and fewer steps are required. This can be used to test specific antibody-to-antigen reactions, and helps to eliminate cross-reactivity between other antibodies.
gnal of the antigen.
The image at the bottom includes the use of a secondary antibody conjugated to an enzyme, though, in the technical sense, this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized. Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement, simplifying the assay, and increasing the specificity and the sensitivity of the assay.
In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interactionELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins.The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).
TYPES
Direct ELISA, Simple and Time-Saving
Initially in a direct ELISA test which is considered to be the simplest type of ELISA the antigen is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block all the other binding sites. While an enzyme is linked to an antibody in a separate reaction, the enzyme-antibody complex is applied to adsorb to the antigen. After excess enzyme-antibody complex is washed off, enzyme-antibody bound to antigen is left. By adding in the enzyme's substrate, the enzyme is detected illustrating the siDirect ELISA, when compared to other forms of ELISA testing, is performed faster because only one antibody is being used and fewer steps are required. This can be used to test specific antibody-to-antigen reactions, and helps to eliminate cross-reactivity between other antibodies.Disadvantages of direct ELISA
The primary antibody must be labeled individually, which can be time-consuming and inflexible when performing multiple experiments. Also, the signal is less amplified in direct ELISA, which means a lower sensitivity and could be viewed as a disadvantage to some
Indirect ELISA, conventional but efficient
Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation with the secondary antibody. However, this may lead to nonspecific signals because of cross-reaction that the secondary antibody may bring about.
1. Micro-well plates are incubated with antigens, washed up and blocked with BSA.
2. Samples with antibodies are added and washed.
3. Enzyme linked secondary antibody are added and washed.
4. A substrate is added, and enzymes on the antibody elicit a chromogenic or fluorescent signal.
Indirect ELISA advantages
- High sensitivity: More than one labeled antibody is bound per antigen molecule;
- Flexible: Different primary detection antibodies can be used with a single labeled secondary antibody;
- Cost-saving: Fewer labeled antibodies are required.
Sandwich ELISA, Highly Sensitive
Sandwich ELISA is a less common variant of ELISA, but is highly efficient in sample antigen detection. Moreover, many commercial ELISA pair sets are built on this sanwich ELISA.
The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least two antibodies act in the sandwich. Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in Sandwich ELISA systems. Monoclonal antibodies recognize a single epitope that allows fine detection and quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. The advantage of Sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive than direct orindirect ELISA), but lower than ELISpot.
Sandwich ELISA procedures can be difficult to optimize and tested match pair antibodies should be used. This ensures the antibodies are detecting different epitopes on the target protein so they do not interfere with the other antibody binding.
The steps are as follows:
- Prepare a surface to which a known quantity of capture antibody is bound.
- Block any nonspecific binding sites on the surface.
- Apply the antigen-containing sample to the plate.
- Wash the plate, so that unbound antigen is removed.
- A specific antibody is added, and binds to antigen (hence the 'sandwich': the Ag is stuck between two antibodies);
- Apply enzyme-linked secondary antibodies as detection antibodies that also bind specifically to the antibody's Fc region (non-specific).
- Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
- Apply a chemical that is converted by the enzyme into a color or fluorescent or electrochemical signal.
- Measure the absorbency or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.
The image at the bottom includes the use of a secondary antibody conjugated to an enzyme, though, in the technical sense, this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized. Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement, simplifying the assay, and increasing the specificity and the sensitivity of the assay.Sandwich ELISA Schematic Procedure:
(1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
Sandwich ELISA advantages:
- High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected
- Suitable for complex samples, since the antigen does not require purification prior to measurement
- Flexibility and sensitivity, since both direct and indirect detection methods can be used
Competitive ELISA: Basic Principles
The central event of competitive ELISA is a competitive binding process executed by original antigen (sample antigen) and add-in antigen. The procedures of competitive ELISA are different in some respects compared with Indirect ELISA, Sandwich ELISA and Direct ELISA. A simplized procedure list is as follow:
- Primary antibody (unlabeled) is incubated with sample antigen.
- Antibody-antigen complexes are then added to 96-well plates which are pre-coated with the same antigen.
- Unbound antibody is removed by washing the plate. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
- The secondary antibody that is specific to the primary antibody and conjugated with an enzyme is added.
- A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the sample antigen concentrati
on, the weaker the eventual signal. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.
on, the weaker the eventual signal. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.
(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample the less labeled antigen is retained in the well and the weaker the signal).
It is common that the antigen is not first positioned in the well.
Competitive ELISA advantages:
- High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected
- Suitable for complex samples, since the antigen does not require purification prior to measurement
- Flexibility and sensitivity, since both direct and indirect detection methods can be used
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