Immunoassays labelled

Fig. 16. The principal of the immunoassay. Labeled antigen (in this case 125I-labeled antigen) competes with unlabeled antigen for binding sites on a set concentration of antibody. Adapted from Ref. (200).

Howanitz, J. H. (1992). Overview of Nonisotopic Immunoassay Labels in Immunochemical Assays and Biosensor Technology for the 1990s, Yasushi Kasahara Robert M. Nakamura, Garry A. Rechnitz (Ed.), Amer Society for Microbiology, pp. 22-35. 

In order to be used as an immunoassay label, an elec-trochemically active compound has to possess suitable electrochemical properties. It has to be soluble in aqueous media and should be stable in solution over a wide pH range. To be detectable, it must allow highly selective electrochemical detection or possess chemical properties to allow selective membranes to be used in the measurement electrode. 

Chemiluminescent labels may be employed in sandwich or competitive antigen assays. In sandwich assays, a solid support holds a primary antibody, and incubation with ligand yields a species that is detectable following a second incubation step with a labeled second antibody. Luminol has been tested as an immunoassay label it may be coupled to proteins through its primary amino group. Luminol reacts with hydrogen peroxide and hydroxide in a microperoxidase-catalyzed reaction, which yields light at 430 nm (Eq. 6.8) ... 

Enzymes are currently the most widely used and investigated labels for immunoassays, because a single enzyme label can provide multiple copies of detectable species. This catalytic amplification results in immunoassay detection limits that rival those of radioimmunoassay without the storage and disposal problems associated with radioisotopes. Enzyme immunoassays label either ligands or antibodies with enzyme, and enzyme activity in bound or free fractions is measured. Heterogeneous immunoassays employing enzymatic labels have been named enzyme-linked immunosorbent assays (ELISAs). ELISA methods usually employ antibody immobilized onto the wells of polystyrene microtiter plates, and may be... 

From the list of immunoassay labels given in Table 6.1, classify each label type as giving either (a) a single detectable event, (b) a continuous signal, or (c) catalytic signal amplification. 

TABLE 9-2 Detection Limits for Isotopic and Nonisotopic Immunoassay Labels ... 

Numerous applications involve coupling liquid-phase chemiluminescence detection to physical or chemical separation processes. Conversely, adequate selectivity can also be achieved for particular analytes in a range of sample matrices through a judicious selection of reagent and reaction conditions. Successful detection strategies have been employed for HPLC, flow analysis, electrophoresis, immunoassay labels, DNA probes, and enzyme reactions. 

Figure 5 Competitive binding immunoassay—labelled antibody.

The microplate model has often been used as a starting platform for electrochemical array development. In a report from Tang et al. [14], a multichannel array consisting of eight channels of Pt electrodes, fitted to the dimensions of standard microplate wells, has been developed, with the required multichannel potentiostat for amperometric detection of the product of an enzymatic immunoassay label. Alkaline phosphatase was used as the model immunoassay label in a model rabbit/anti-rabbit IgG study, where the second IgG is labeled with enzyme. The enzyme product provided the amperometric signal with a detection limit in the low pM range. 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Enzyme Immunoassay. In EIA, antibody (or antigen) is labeled with (or conjugated to) an enzyme, and this reagent is used to complex and quantify the target antigen (or antibody) in a sample. Conjugation may utilize a variety of chemical methods. 

In the most common method for chemiluminescent immunoassay (GLIA), after the immunological reaction and any necessary separation steps, the labeled compounds or complexes react with an oxidizer, eg, hydrogen peroxide, and an enzyme, eg, peroxidase, or a chelating agent such as hemin or metal... 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

There should be specific, saturable binding to the receptor, accompanied by pharmacological characteristics appropriate to the functional effects, demonstrable using a radioactive, eg, tritium or iodine-125, ligand to label the receptor. Radioligand binding assays (1,6) have become a significant means by which to identify and characterize receptors and enzymes (see Immunoassays Radioactive tracers). Isolation of the receptor or expression of the receptor in another cell, eg, an oocyte can be used to confirm the existence of a discrete entity. 

Radioisotopes have become very important ia the practice of modem medicine, for both diagnosis and treatment. Some diagnoses are done by injecting a radionucHde ia a biochemical form such that it goes to a particular organ, and the measured radiation then allows the functional level of that organ to be determined. A common treatment is to expose a portion of the body, for example a tumor, to radiation from a radioisotope with the source either internal or external to the body. Another usage iavolves radioactively labeled antibodies (see Immunoassay). 

The immunochemical interaction between the antigen and antibody is very specific. By labeling either the antigen or antibody, the method s sensitivity is increased. The most frequently used labels to increase sensitivity are radionucHdes (see Radioisotopes) where the assay process is called radioimmunoassay (RIA), or en2ymes where the assay is named en2yme immunoassay (ElA) (see Enzyme applications). 

Immunochemical metliods drat utilize radioisotopic labeling can detect tire use of anabolic sex hormones drat increase die growdi in meat animals. Stilbene [588-59-0] trenbolone [10161 -33-8] and zeranol [55331-29-8] C gH2 0, can be successfully monitored by diese immunoassay techniques... 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

---