9- Labeled Immunoassays Fluorescent & Chemiluminescent Immunoassays In 1944 it was demonstrated that antibodies could be labeled with molecules that fluoresce. These fluorescent compounds are called fluorophores or fluorochromes. They have the ability to absorb energy from an incident light and convert that energy into light of a longer wavelength and lower energy as the excited electrons return to the ground state. Fluorophores are typically organic molecules with a ring structure, and each has a characteristic optimum absorption range. The time interval between absorption of energy and emission of fluorescence is very short and can be measured in nanoseconds. Ideally, a fluorescent molecule should exhibit high intensity, which can be distinguished easily from background fluorescence. It should also be stable and have a high molar extinction coefficient ( a measurement of how strongly a chemical species absorbs light at a given wavelength). The two compounds most often used are fluorescein and rhodamine, because these can be readily coupled with antigen or antibody. Fluorescein absorbs maximally at 490 to 495 nm and emits a green color at 517 nm. It has a high intensity, good photostability, and a high quantum yield. Tetramethylrhodamine absorbs at 550 nm and emits red light at 580 to 585 nm. Because their absorbance and emission patterns differ, fluorescein and rhodamine can be used together. Newer compounds that are beginning to be used are phycobiliproteins derived from algae, porphyrins and chlorophylls, all of which exhibit red fluorescence at over 600 nm. Heterogeneous Fluorescent Immunoassays Heterogeneous assays, which require a separation step, include the following: indirect, competitive and sandwich assays. These are based on the same principle as those of enzyme immunoassays, but in this case the label is fluorescent. Such label can be applied to either antigen or antibody.
Use of solid phase is the typical means of separation in heterogeneous assays. Microbeads made of polysaccharides and polyacrylamides have been used by a number of manufacturers. Either antigen or antibody can be attached to the beads and reacted with analyte and a fluorescent labeled analyte. Then reaction mixture is centrifuged, the supernatant is discarded, and the beads are analyzed for fluorescence. Homogenous Assays Homogenous FIA requires no separation of procedure, so it is rapid and simple to perform. There is only one incubation step and no wash step, and usually competitive binding is involved. The basis for this technique is the change that occurs in the fluorescent label on antigen when it binds to specific antibody. Such changes can be related to wavelength emission, or polarity. There is a direct relationship between the amount of fluorescence measured and the amount of antigen in the patient sample. As binding of patient antigen increases, binding of the fluorescent analyte decreases and hence more fluorescence is observed. Typically homogenous assays including enzyme assays have suffered from low sensitivity. Hence, most research has aimed at increasing sensitivity and newer procedures have been developed that include florescence polarization immunoassay (FPIA), florescence excitation transfer immunoassay, and time resolved fluorescence immunoassay. All of these require specific instrumentation. Fluorescence Polarization Immunoassay (FPIA) It is based on the change of polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody. Incident light directed at the specimen is polarized with a lens or prism so the waves are aligned in one plane. If a molecule is small and rotates quickly enough, when it is excited by polarized light, the emitted light is unpolarized. If however the labeled molecule is bound to antibody, the molecule is unable to tumble as rapidly, and it emits an increased amount of polarized light. Thus the degree of polarized light reflects the amount of labeled analyte that is bound.
In FPIA, labeled antigens compete with unlabeled antigen in the patient sample for a limited number of antibody binding sites (Figure 9.1). The more the antigen that is present in the sample, the less the fluorescence labeled antigen is bound and the less the polarization that will be detected. Figure 9.1. Competitive fluorescence polarization immunoassay (FPIA). With competitive binding, antigen from the specimen and antigenfluorescein (AgF) labeled reagent compete for binding sites on the antibody. As a homogeneous immunoassay, the reaction is carried out in a single reaction solution, and the bound Ab-AgF complex does not require a wash step to separate it from free labeled AgF. FPIA is utilized to provide accurate and sensitive measurement of small toxicology analytes such as therapeutic drugs, and drugs of abuse, toxicology and some hormones. FPIA utilizes three key concepts to measure specific analytes in a homogeneous format: fluorescence, rotation of molecules in solution, and polarized light. Fluorescence: Fluorescein is a fluorescent label. It absorbs light energy at 490nm and releases this energy at a higher wavelength (520nm) as fluorescent light. Rotation of Molecules in Solution: Larger molecules rotate more slowly in solution than do smaller molecules. This principle can be used to distinguish between the smaller antigen-fluorescein molecule, AgF, which rotates rapidly, and the larger Ab-AgF complexes, which rotate slowly in solution.
Polarized Light: Fluorescence polarization technology distinguishes antigen-fluorescein (AgF) label from antibody bound- antigenfluorescein (Ab-AgF) by their different fluorescence polarization properties when exposed to polarized light (Figure 9.2). Polarized light describes light waves that are only present in a single plane of space. When polarized light is absorbed by the smaller AgF molecule the AgF has the ability to rotate its position in solution rapidly before the light is emitted as fluorescence. The emitted light will be released in a different plane of space from that in which it was absorbed and is therefore called unpolarized light. With the larger sized Ab-AgF complex, the same absorbed polarized light is released as polarized fluorescence because the much larger Ab-AgF complex does not rotate as rapidly in solution. The light is released in the same plane of space as the absorbed light energy, and the detector can measure it (Figure 9.3). Figure 9.2. Detection of fluorescence in fluorescein-conjugated complexes 490nm Figure 9.3. Measurement of large complexes using fluorescence, rotation, and polarized light in FPIA
Measurement of large complexes using fluorescence, rotation, and polarized light in FPIA FPIA results in an inverse dose response curve such that lower levels of patient analyte result in a higher signal (in this case, the signal is polarized light) (Figure 9.4). High signal at low patient analyte levels results in a highly sensitive assay. Advantages and Disadvantages Figure 9.4 FPIA results in lower inverse relationship between signal and concentrate of analyte. Advantages Sensitivity is higher than those of radiolabels and enzyme reactions. The methodology is simple and there is no need to deal with and dispose of hazardous substances. Disadvantages The main problem is the separation of the signal on the label from background fluorescence because of different organic substances normally present in serum. Nonspecific binding to substances in serum can cause diminishing of the signal and change the amount of fluorescence generated. Any bilirubin or hemoglobin present can absorb either the excitation or emission energy. It requires expensive dedicated instrumentation, which may limit its use in smaller laboratories.
Chemiluminescent Immunoassays Several recently developed immunoassays use the principle of chemiluminescence to follow antigen antibody combination. Chemiluminescence is the emission of light caused by a chemical reaction producing an excited molecule that decays back to its original ground state. A large number of molecules are capable of chemiluminescence, but some of the most common substances used are luminol, acridium esters, peroxyoxalates, ruthenium derivative and dioxetanes (Figure 9.5). When these substances are oxidized, typically using hydrogen peroxide and an enzyme for a catalyte, intermediates are produced that are of a higher energy state. These intermediates spontaneously return to their original state, giving off energy in the form of light (Figure 9.6). Light emissions range from a rapid flash of light to a more continuous glow that can last for hours. Different types of instrumentation are necessary for each kind of emission. Figure 9.5. Detection of HRP using a luminol-based chemiluminescent substrate. Figure 9.6. Signal production mechanisms in chemiluminescent substrate.
Advantages and Disadvantages Advantages Have an excellent sensitivity comparable to EIA and RIA. Reagents are stable and relatively nontoxic. The sensitivity of some assays has been reported to be in the range of attamoles (10-18 ) to zeptomoles ( 10-21 ). Because very little reagent is used, they are inexpensive to perform. Detection systems basically consist of photomultiplier tubes which are simple and relatively inexpensive Disadvantages False results may be obtained if there is lack of precision in injection of the hydrogen peroxide If some biological materials such as urine or plasma cause diminishing of the light emission.