Immunochemical methods using specific

Specific quantitative assay of particular proteins by immunochemical methods using specific antisera and measurement of the antigen-antibody (Ag-Ab) complexes by nephelometry, turbidimetry, radial immunodiffusion (RID) or radioimmunoassay (RIA), or enzyme immunoassay. 

Immunochemical methods using specific albumin antiserum. Radial immunodiffusion, electroimmunodiffusion and nephelometric techniques can all be used. 

Since antibody probes are extremely sensitive to variations, even subtle ones, in protein conformation, they are potent but rather delicate tools for the study of protein folding or unfolding. For the first time, Anfinsen s group developed a quantitative immunochemical approach to study the refolding of staphylococcal nuclease (Sachs et aL, 1972a,b,c, 1974 Eastlake et ai, 1974 Furie et aL, 1974, 1975). From that, different methods using specific antibodies for detection and characterization of intermediates in protein... 

Va.ria.tions in Methods. The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be cmde antisemm, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is mn, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

The concept of immunoassay was first described in 1945 when Landsteiner suggested that antibodies could bind selectively to small molecules (haptens) when they were conjugated to a larger carrier molecule. This hapten-specific concept was explored by Yalow and Berson in the late 1950s, and resulted in an immunoassay that was applied to insulin monitoring in humans. This pioneering work set the stage for the rapid advancement of immunochemical methods for clinical use. 

Immunochemical methods are valuable because of their sensitivity and specificity. The sensitivity depends on the method used to determine an end point. One of the reaction components may be tagged with radioactivity, or tagged by covalent binding of an enzyme capable of being detected, or by covalent binding of a totally unrelated species (i.e., fluorescein). 

Problems may also arise with respect to specificity when ELISAs are applied for trace residue analysis, because any compound whose molecule is in part identical with or closely similar to the antigenic determinant of the analyte can compete for antibody-binding sites. Therefore, immunochemical methods are valuable for screening and testing purposes but cannot be considered as definitive from a regulatory perspective. For legal enforcement use, these methods should be used as part of an analytical system that consists of additional methods capable of definitively identifying the compounds of interest. 

Although both polyclonal and monoclonal antibodies have been effectively used in immunochemical assays, only the latter can provide the high specificity required in some applications. Antibody specificity, on the other hand, is both a major advantage and disadvantage for immunochemical methods. It allows for highly selective detection of analytes but at the same time may complicate the development of multiresidue methods. Moreover, production of monoclonal antibodies requires special expertise and it is much more expensive than polyclonal antibodies. Thus, in cases where a range of analytes similar in molecular structure are required to be determined, a polyclonal may be more suitable than a monoclonal antibody. 

A series of specific inhibitors of trimming reactions, some of whose structures are shown in Fig. 20-7, has provided important insights 253-255 Use of these inhibitors, together with immunochemical methods and study of yeast mutants,250 252 256 is enabling us to learn many details of glycoprotein biosynthesis. 

For mycotoxin analyses radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISAs) and affinity chromatography are the principal immunochemical methods in commercial application. Immunoaffinity columns or cartridges for specific mycotoxins are now being increasingly used in preliminary clean-up of extracts prior to final analysis by HPLC or GLC methods. 

Also, HPLC methods with electrochemical or fluorescent detection are used (H19, M3). In proteins, dityrosine can be estimated by immunochemical methods employing dityrosine-specific antibodies (K5). Measurements of o,o -dityrosine and o-tyrosine levels in rat urine express dityrosine contents in skeletal muscle proteins, and have been proposed as the noninvasive oxidative stress test in vivo. One should be aware, however, that A-formylkynurenine, also formed in protein oxidation, has similar fluorescence properties as dityrosine (excitation 325 nm, emission at 400-450 nm) (G29). Also, oxidation of mellitin when excited at 325 nm produces an increase in fluorescence at 400—450 nm, despite the fact that mellitin does not contain tyrosine. Oxidation of noncontaining Trp residues ribonuclease A and bovine pancreatic trypsin inhibitor with "OH produces loss of tyrosine residues with no increase in fluorescence at 410 nm (S51). There are also methods measuring the increased hydrophobicity of oxidized proteins. Assays are carried out measuring protein binding of a fluorescent probe, 8-anilino-l-naphthalene-sulfonic acid (ANS). Increase in probe binding reflects increased surface hydrophobicity (C7). 

The specific activity of proteins assayed by direct immunochemical methods or those used as standards in radioimmunoassays profoundly affects the resolution attainable by these techniques. Therefore, the nature of the radioactive label to be used in such experiments must be considered carefully. Table 8-6 lists the isotopes available for this purpose along with the number of atoms of each isotope that must be incorporated to produce an arbitrary counting rate. As can be seen here, 557 atoms of H and 261,672 atoms of C must be incorporated into every molecule of protein to yield the same number of disintegrations per minute as only one I or 11 S molecules. S-methionine is often the isotope of choice for many direct immunochemical procedures since it is relatively inexpensive to prepare at high specific activity. On the other hand, the relative ease with which radioactive iodine may be incorporated into a purified antigen makes it the isotope of choice for radioimmunoassay methods. Of the two iodine isotopes available, is most often used because of its longer half-life. This is an important consideration since it usually takes more than 1 week to prepare and test a labeled antigen prior to its experimental use. 

Despite the fact that immunoassays can be sensitive, specific and rapid, their application to detect compounds of environmental importance has been limited to relatively few laboratories (7-10). This symposium volume serves an important function since it focuses on the current status of the immunochemical methods being used for environmental analyses. Many of the accompanying papers consider the basic principles and essential steps in setting up immunoassays, i.e. the covalent linkage of small molecules to carrier proteins, immunization with these... 

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