Welcome to Badrilla, a reagents company with antibodies suitable for ELISA formats (phospho-specific targets, epitope tags, fluorescent proteins). Please brouse our site to see which antibodies might assist your research.
Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a popular form of immunoassay which can analyse large numbers of samples for the same biomarker (or analyte) rapidly and easily. This is a great advantage over other immunoassays, however researchers need to be convinced of the specificity of the assay - so that correct interpretation of the resuts can be drawn. There are a number of different forms of ELISA:
(i) indirect ELISA - where the biomarker is immobiised on a surface and the binding of an antibody to that target is measured. Indirect ELISA is fast and easy to establish, but it can have low specificity as it relies on the properties of one antibody. Research needs to be performed to check that the antibody recognises only the biomarker in the samples being analysed.
(ii) sandwich ELISA - where a "capture" antibody is bound to a surface, a biomarker (or analyte) from a sample is bound by this capture antibody, and a detection antibody to the biomarker also binds to measure the concentration of biomarker in the sample. This is a more strigent form of ELISA, as the binding of two antibodies to the same biomarker is required for signal to be produced. This reduces the likelihood of non-specific signal from non-target proteins.
(iii) competitive ELISA - like the "indirect ELISA" but in the presence of soluble biomarker from the specimen (or sample). Now the antibody can bind to the immobilised biomarker or the soluble biomarker (hence the term competition) and the concentration of biomarker from the sample can be determined by the loss of antibody binding to the immobilised biomarker. The strigency of this assay is the same as indirect ELISA, namely lower strigency.
A number of proptocls for the differnt ELISA assays follow:
The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:
- Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
- The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
- A concentrated solution of non-interacting protein, such as Bovine Serum Albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
- The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
- The plate is washed to remove any unbound detection antibody. After this wash, only the antibody-antigen complexes remain attached to the well.
- Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
- Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
- Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
- View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
A sandwich ELISA. (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.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
- Prepare a surface to which a known quantity of capture antibody is bound.
- Block any non specific binding sites on the surface.
- Apply the antigen-containing sample to the plate.
- Wash the plate, so that unbound antigen is removed.
- Apply primary antibodies that bind specifically to the antigen.
- Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
- Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
- Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
- Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.
The use of a secondary antibody conjugated to an enzyme is a convenient format for detection. It 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. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. 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.
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
- Unlabeled antibody is incubated in the presence of its antigen.
- These bound antibody/antigen complexes are then added to an antigen coated well.
- The plate is washed, so that unbound antibody is removed. (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, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
- A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
We acknowledge authors on wikipedia for the protocols here (http://en.wikipedia.org/wiki/ELISA ; copyleft applies to this section of our site: which signifies that you are free to reproduce the content contained within boxed sections, according to the terms of the Wikipedia foundation: http://en.wikipedia.org/wiki/GNU_Free_Documentation_License).