Immunoassay reagents

Enzymes can be linked to immunoassay reagents to amplify detection by the use of fluorogenic substrates. Enzyme-linked fluoroimmunoassays (ELFIAs) are very similar to photometric EIAs in format and workflow. EIAs are widely used, and many commercial ELFIA assays and systems are available/15 The most commonly used enzymes in ELFIAs are horseradish peroxidase, alkaline phosphatase, and fi-D-... 

A number of different testing kits based on immunoassay technology are available for rapid field determination of certain groups of compounds, such as benzene-toluene-ethylbenzene-xylene (EPA 4030) or polynuclear aromatic hydrocarbons (EPA 4035, Polycyclic Aromatic Hydrocarbons by Immunoassay). The immunoassay screening kits are self-contained portable field kits that include components for sample preparation, instrumentation to read assay results, and immunoassay reagents. 

S168 Drost, R.H., Schultz, H.U., Maes, R.A.A. and van Rossum, J.M. (1987). A comparative study of a monoclonal immunoassay reagent strip test and high pressure liquid chromatography for theophylline. Clin. Toxicol. 25, 231-241. 

M Adamczyk, et al. Immunoassay reagents for psychoactive drugs. Part 3. Removal of phenothiazine interferences in the quantification of tricyclic antidepressants. Ther Drug Monit 15 436, 1993. 

Uric acid, a serum component having a maximal normal level of 6 mg/100 ml (Orten and Neuhaus, 1982), is a major electroactive interference. A hydrodynamic voltammogram for uric acid displayed an i/, of 430 mV versus Ag/AgCl. Consequently potential selection could not be used to resolve NADH from the more easily oxidizable uric acid. Immunoassay reagents were also sources of electrochemical interference. 

Company (Palo Alto, CA). Immunoassay reagents and calibrators were prepared as prescribed in the kit instruction manual. Six digoxin calibrators (0-7.5 ng/ml in human serum) were provided. 

Reagent for enzyme-linked immunoassay Isolation by immunoadsorption of immunoglobulin G Fc fragment-binding glycoproteins from human blood platelets Reagent for enzyme immunoassay Reagent for enzyme immunoassay Purification by immunoadsorption of anti-carbohydrate antibodies... 

To detect the immunoassay reagent, the most popular and effective method is to use an analytical label such as radioactive elements, enzymes, and others. According to the labels, the immunoassay detection approaches can be divided to optical detection method and nonoptical detection method. 

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. 

EIAs can be used per se or with a spectrophotometer. Traditionally, EIAs have been developed in 96-weU microtiter plates which provide the immobilization support for the assay, the reaction vessel, and, when linked to a spectrophotometer-based reader, a rapid means to detect and quantify the color resulting from interaction of a substrate with the antibody—antigen—enzyme complex. Automated immunoassay analyzers targeted primarily for use in the clinical laboratory have taken automation one step further, utilizing robotics to carry out all reagent additions, washings, and final quantification including report preparation. 

Fluorescence Immunoassay. Basic FIA follows the same formats and approaches as EIA. The difference Hes in the indicator a fluotophote is used instead of an enzyme. This allows direct quantification of the indicatot—antibody—antigen complex, or free indicator-reagent, without the need for a substrate. 

The advantages of homogenous immunoassays are simple formats and rapid data output producing user-friendly and cost-effective products. Technical challenges to consider, however, are the necessity to remove or minimize background interference from the reagents and nonspecific binding reactions. 

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). 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

See Immunoassay Medical diagnostic reagents Radioactive elements. 

Immunoassays. Immunoassays (qv) maybe simply defined as analytical techniques that use antibodies or antibody-related reagents for selective deterrnination of sample components (94). These make up some of the most powerflil and widespread techniques used in clinical chemistry. The main advantages of immunoassays are high selectivity, low limits of detection, and adaptibiUty for use in detecting most compounds of clinical interest. Because of their high selectivity, immunoassays can often be used even for complex samples such as urine or blood, with Httle or no sample preparation. 

See Automated INSTRUMENTATION Immunoassay Medicad diagnostic reagents. 

Fig. 13. General protocol for heterogeneous enzyme immunoassay preparation of reagent cuvettes by coating Ab, competitive assay format, and sandwich assay format. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem, 1985, 57, 1321A. Copyright 1985, American Chemical Society)...

Fig. 10. Comparison of a typical homogeneous immunoassay (left) with a heterogeneous format (right). Depending on choice of reagents, there are many possible variations on these basic schemes...

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

EIAs are more desirable for the measurement of agrochemicals than enzyme-linked immunosorbent assays (ELISAs) for several reasons. EIAs are easier to run, require minimal liquid transfers, and are completed in brief time frames, approximately 40 min for tube assays to 2.5 h for microtiter plate assays. In contrast, ELISAs are more complex, have many steps involving transfer of reagents, and require 6-8 h to complete. Most commercially available immunoassays utilize the EIA format. 

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