What Types Of ELISAs Are There?
The steps of "indirect" ELISA follows the mechanism below:
- A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.
- A solution of nonreacting protein, such as bovine serum albumin or casein, is added to block any plastic surface in the well that remains uncoated by the antigen.
- The primary antibody is added, which binds specifically to the test antigen coating the well. This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen.
- A secondary antibody is added, which will bind the primary antibody. This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody.
- A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows the secondary antibody has bound to primary antibody, which strongly implies the donor has had an immune reaction to the test antigen. This can be helpful in a clinical setting, and in research.
- The higher the concentration of the primary antibody present in the serum, the stronger the color change. Often, a spectrometer is used to give quantitative values for color strength.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. Within common-sense limitations, the enzyme can go on producing color indefinitely, but the more primary antibody is present in the donor serum, the more secondary antibody + enzyme will bind, and the faster the color will develop. A major disadvantage of the indirect ELISA is the method of antigen immobilization is not specific; when serum is used as the source of test antigen, all proteins in the sample may 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 or direct ELISA provides a solution to this problem, by using a "capture" antibody specific for the test antigen to pull it out of the serum's molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) 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 (error inherent in a test) is often used to distinguish positive from negative samples. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule. For example, if a test sample returns an OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same analyte concentration as the sample.
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, a "sandwich" ELISA, is used to detect sample antigen. The steps are:
- A surface is prepared to which a known quantity of capture antibody is bound.
- Any nonspecific binding sites on the surface are blocked.
- The antigen-containing sample is applied to the plate.
- The plate is washed to remove unbound antigen.
- A specific antibody is added, and binds to antigen (hence the 'sandwich': the Ag is stuck between two antibodies)
- Enzyme-linked secondary antibodies are applied as detection antibodies that also bind specifically to the antibody's Fc region (nonspecific).
- The plate is washed to remove the unbound antibody-enzyme conjugates.
- A chemical is added to be converted by the enzyme into a color or fluorescent or electrochemical signal.
- The absorbency or fluorescence or electrochemical signal (e.g., current) of the plate wells is measured to determine the presence and quantity of antigen.
The image to the right 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.
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples:
- Unlabeled antibody is incubated in the presence of its antigen (sample).
- These bound antibody/antigen complexes are then added to an antigen-coated well.
- The plate is washed, so 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.
- The reaction is stopped to prevent eventual saturation of the signal.
Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the more labeled antigen is retained in the well and the stronger the signal.
Commonly, the antigen is not first positioned in the well.
For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV antigen. Two specific antibodies are used, one conjugated with enzyme and the other present in serum (if serum is positive for the antibody). Cumulative competition occurs between the two antibodies for the same antigen, causing a stronger signal to be seen. Sera to be tested are added to these wells and incubated at 37°C, and then washed. If antibodies are present, the antigen-antibody reaction occurs. No antigen is left for the enzyme-labelled specific HIV antibodies. These antibodies remain free upon addition and are washed off during washing. Substrate is added, but there is no enzyme to act on it, so positive result shows no color change.