Immunoassay specificity

Immunoassay specificity and selectivity are strictly dependent on the antibodies used. A good knowledge of the target allows to build up a good immunoassay design. 

Figure 1. Illustration of importance of hapten selection on immunoassay specificity.

This chapter is designed to review some of the approaches now used for automating immunoassays, specifically the radioimmunoassay (RIA) and the enzyme-linked immunosorbent assay (ELISA), and to describe the use of magnetic devices for processing solid phase elements in these assays. 

J. J. Pratt, M. G. Woldring, R, Boonman, and J. Kittikool, Specificity of immunoassays. II. Heterogeneity of specificity of antibodies and antisera used for steroid immunoassay and the selective blocking of less specific antibodies, including a new method for the measurement of immunoassay specificity, Eur.. Nuc. Med., 4 159-170 (1979). 

When immunoassay specificity has not been inherent in the antibody employed in the assay, separation steps such as high performance liquid chromatography have... 

Abdel-Hamid et al. [122] used a flow-injection amperometric immunofll-tration assay system for the rapid detection of total E. coli and Salmonella. Disposable porous nylon membranes served as a support for the immobilization of anti- ]. coli or anti-Salmonella antibodies. The assay system consists of a flow-injection system, a disposable filter-membrane, and an amperometric sensor. A sandwich immunoassay specifically and directly detected 50 cells ml total E. coli or 50 cells ml Salmonella. The immunosensor can be used as a highly sensitive and automated bioanalytical device for the rapid quantitative detection of bacteria in food and water. 

Specificity of an immunoassay is usually measured by determining the extent that compounds that are structurally similar to the test analyte react in the assay. The determination of the assay reactivity of an array of potential cross-reactant is routinely performed in immunoassay development. A panel of suspect cross-reactant, should be selected on the basis of structural similarities to the test analyte and on the expected occurrence along with the test analyte in the sample. Thus, an immunoassay specific for 17/1-estradiol should be tested for reactivity with estrone, estriol, 17a-estraiol and testosterone. Other compound testing might also be indicated. 

The majority of immunoassays for speciation are used to analyze raw, cooked, or otherwise processed meats from terrestrial, commercially raised livestock (beef, pork, poultry, sheep, horse, and deer). Early speciation tests worked only in raw, unprocessed samples. Over time, however, tests have been developed that can speciate cooked meats as well as highly processed products such as meat and bone meals. In this section we give a detailed explanation of the development and use of immunoassays specific to this area of speciation testing. 

Sensitive immunoassays specific for PAH-DNA adducts allow the detection of one adduct per 10 nucleotides. Fluorimetry has also been used as an alternative detection method to immunoassays. In another method, isomeric tetrols of PAH are liberated by acid hydrolysis of the DNA-PAH adducts and analyzed by LC with fluorescence detection. Structural studies and detailed characterization of PAH metabolites and their conjugates has been performed by trapping the corresponding fractions at the exit of the LC system. As an example, the total characterization of the in t /tro-formed benzo(a)pyrenetetra-hydrodiol-epoxide-guanosine adduct has been achieved by a combination of nuclear magnetic resonance, circular dichroism, and mass spectroscopic techniques. 

Immunoassays, specific protein-binding assays, and radioisotope tests are sometimes used for the determination of water-soluble vitamins. These are the only feasible and practical methods for the quantification of certain vitamins in physiological samples. The principle of competitive protein binding (CPB) using labeled radioactive or fluorescent tracer is still routinely applied to quantification of serum vitamin... 

The levels of P-D-2-acetamido-2-deoxyhexosidases A and B in the tissues and body fluids of normal individuals and Tay-Sachs patients have been measured by sensitive immunoassays specifically developed for this purpose. The results showed that normal tissues contain comparable amounts of both isoenzymes, whereas the tissues of Tay-Sachs patients contain neither the A isoenzyme nor material that carries A-specific antigenic determinants. The use of these assays in the diagnosis of Tay-Sachs disease was discussed. 

Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

The specific enzyme to be used in an EIA is deterrnined according to a number of parameters including enzyme activity and stabiUty (before, during, and after conjugation), cost and availabiUty of the enzyme substrate, and the desired end point of the EIA, such as color. Most EIAs utilize a colored end point which can be readily deterrnined both visually and spectrophotometricaHy. Table 1 Hsts a number of enzymes which have been used in immunoassays and their substrates. 

Chemiluminescent Immunoassay. Chemiluminescence is the emission of visible light resulting from a chemical reaction. The majority of such reactions are oxidations, using oxygen or peroxides, and among the first chemicals studied for chemiluminescence were luminol (5-amino-2,3-dihydro-l,4-phthalazinedione [521-31-3]) and its derivatives (see Luminescent materials, chemiluminescence). Luminol or isoluminol can be directly linked to antibodies and used in a system with peroxidase to detect specific antigens. One of the first appHcations of this approach was for the detection of biotin (31). 

The singularity of MAbs and the ease of mass production appeared to be the answer to rapid development of highly specific immunoassays. Companies were formed to produce MAbs and incorporate them into assays. In fact, such assays have been developed and have proved very successful for infectious diseases, hormones, and other clinical analytes. 

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. 

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

Lanthanide luminescence apphcations have already reached industrial levels of consumption. Additionally, the strongly specific nature of the rare-earths energy emissions has also led to extensive work in several areas such as photostimulable phosphors, lasers (qv), dosimetry, and fluorescent immunoassay (qv) (33). 

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. 

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. 

Radiotracers have also been used extensively for the quantitative rnicrodeterrnination of blood semm levels of hormones (qv), proteins, neurotransmitters, and other physiologically important compounds. Radioimmunoassay, which involves the competition of a known quantity of radiolabeled tracer, usually I or H, with the unknown quantity of semm component for binding to a specific antibody that has been raised against the component to be deterrnined, is used in the rnicro deterrnination of physiologically active materials in biological samples (see Immunoassay). 

Biopolymers are employed in many immunological techniques, including the analysis of food, clinical samples, pesticides, and in other areas of analytical chemistry. Immunoassays (qv) are specific, sensitive, relatively easy to perform, and usually inexpensive. For repetitive analyses, immunoassays compare very favorably with many conventional methods in terms of both sensitivity and limits of detection. 

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