Protein immunochemical methods

Iodine is incorporated in thyroid proteins to form thyroxin and 3-I-thyroxine, both hormones essential for life. They are determined by immunochemical methods. Deficiency of I may lead to crop disease. 

The Use of Immunochemical Methods in Studies on Proteins Pierre Grabar... 

Adequate removal of vector, host cell culture medium and reagent-derived contaminants from the product. The reduction capacity for DNA is established by spiking. The reduction of proteins of animal origin can be determined by immunochemical methods. 

Key to the successful production of generic antisera for the tetracycline antibiotics has been the synthesis of the hapten 4-hydrazino-4-dedimethylamino-tetracycline and its subsequent conjugation to protein (73). The immunochemical method developed from this sera could detect tetracycline, chlortetracycline, and oxytetracycline residues in meat and milk, with adequate sensitivity (19). 

Al any methods used to study protein interactions and proteolysis in - - complex mixtures are often unsuitable for application to food systems, where severe heat treatments or enzymic modification may change the properties of proteins and their reactivity in an analytical procedure. Chromatographic, electrophoretic, and immunochemical methods are excellent tools for fractionating protein mixtures and measuring the concentrations of the individual components, but heat treatment or enzymic... 

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 original experiments on PDI activity were carried out with microsomal membranes, suggesting the localization of this protein in the endoplasmic reticulum. This has since been conhrmed both by subcellular localization studies (Lambert and Freedman, 1985) and by immunochemical methods (Koch, 1987). However, these studies revealed that PDI does not behave like a typical ER membrane protein. Indeed, for some time there was a question as to whether there was more than one form of PDI due to the presence of this protein in the cytosol. This has now been discounted by studies on the latency of PDI that show that if microsomes are prepared carefully then the activity is entirely latent (Lambert and Freedman, 1985). Thus, although PDI exists as an ER protein, it is easily released from microsomes by freeze thawing, mild alkaline treatment, detergents, or sonication, suggesting that PDI is either a soluble protein or is only loosely associated with the ER membrane. 

The advantage of iron labeling for electron microscopy has inspired the use of some other metals and iron in the form of other compounds, for instance ferrocene, e.g. bis-pentadienyl iron Mercury uranium and osmium are also widely used in electron microscopy for protein labeling. Because they are easy to detect and because they attach themselves readily to antibodies and antigens, metals are very promising labels in immunochemical methods. Voller forecasts the development of a new generation of simple metaloimmunoassays. 

Apart from assays of proteolytic activity and immunochemical methods (7.1 and 7.5), protein labeling is exploited in other areas of analysis. Noteworthy among them are protein determination in complex mixtures or even directly in cells, protein separation (affinity chromatography and electrophoresis) and protein detection following various separation techniques. For this purpose different labels are applied. 

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. 

Five, genetically distinct, /8-D-fructofuranosidases have been described for strains of Saccharomyces hybrids. The behavior and kinetics of each enzyme are very similar.362,451 W. L. Smith and Ballou have purified the mannan-protein /3-D-fructofiiranosidases of three strains of Saccharomyces cerevisiae whose cell walls have differences in mannan structure.452 By use of immunochemical methods, they found that the structure of each /3-D-fructofuranosidase mannan is similar to that of the cell wall of the corresponding strain only. Mutations affecting the structure of the one also produced similar changes in the other. 

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... 

The adaptation of immunochemical methods for many classes of small compounds has made it possible to develop assays for pesticide residue detection. A cELISA was developed for methoprene, an analog of insect juvenile hormone. Because of its size, methoprene does not elicit an immune response by itself. However, by conjugating methoprene to a carrier protein it was made immunogenic in animals. A four-carbon spacer group was incorporated between methoprene and the carrier protein. The spacer was first coupled to methoprene acid by a series of protection/deprotection steps. 

This technique has gained widespread acceptance as an immunochemical method for identifying proteins. As in lEP and CRIE, a first-dimension electrophoresis is performed in agarose gel to separate the proteins in the mixture. 

IgA, with these variant albumins varies with storage conditions, pH, and concentration of low molecular weight —SH compounds, such as glutathione hence the same serum sample may appear different on electrophoresis at different times. Perhaps the most important laboratory consequence of these variants is the formation of complexes with AAT, resulting in apparent deficiency of this protein on electrophoretic patterns. However, quantification of AAT by immunochemical methods is normal. 

TTR migrates anodal to albumin on routine serum electrophoresis the presence of a TTR band is considered one marker of good quality electrophoresis. However, levels can be only roughly semiquantified from the intensity of the band. RBP dissociates during electrophoresis and migrates anodal to transferrin, but the band is usually too faint to see. Either protein can be quantified by routine immunochemical methods. 

Specific quantitative assays of particular proteins by immunochemical methods (see Chapter 9) using specific antisera and measurement of the antigen antibody complexes by nephelometry, turbidimetry, RID, or electroim-muno assay or, if present in very low concentrations, by RIA, enzyme immunoassay (EIA), fluorescence immunoassay, or chemiluminescence. 

Most immunochemical methods are applicable to the measurement of any of the proteins in this chapter (see Chapter 9). Because of their speed and ease, nephelometric and tur-bidimetric methods are most widely used for most serum proteins. These techniques are performed either by measuring the amount of Ag-Ab complex formation (equhibrium methods) or by measuring the rate of complex formation (kinetic methods). The kinetic methods are slightly faster, with measurements completed within 20 s however, kinetic assays are somewhat less sensitive because low-affinity antibodies do not have time to react. In addition, many kinetic methods obtain the baseline reading after addition of antiserum, which can reduce the measured signal with high-affinity antibodies. A compromise is often used, with timed measurement before true equilibrium. 

In biochemistry, non-enzymatic proteins are analyzed by immunochemical methods. Especially the popular enzyme-linked immunosorbent assay (ELISA)... 

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