Using Immunological Methods

Immunoprecipitation methods may be employed in conjunction with electrophoretic separations in at least two ways In the first, specific antibodies or mixtures of them are used to form precipitation arcs with the individual electrophoretically separated proteins. As the method is ordinarily employed in immunoelectrophoresis, small differences in electrophoretic mobility are difficult to detect. However, modifications have been developed which make the method more precise. For example, chimpanzee transferrin, haptoglobin, and a2-niacroglobulin have mobilities different from their human homologues. When human and chimpanzee sera are run in parallel, and antiserum is diffused in from both sides and allowed to form complete ovals around the antigens, these mobility differences are readily observed (Fig. 3), as shown by Williams and Wemyss (1961). Methods for increasing the resolution of this system are now under development in the MAN Program laboratories at Oak Ridge and may solve the problem. 

The pattern seen in Fig. 3 is complex and may be much simplified by including only antibodies against a selected subset of proteins. Alternatively, one or more different fluorescent labels may be attached to selected antibodies, allowing their unambiguous identification. 

The second general method is to use antibodies to precipitate enzymes which are then identified by the techniques of enzyme histochemistry. A 

TABLE 3. Enzymes Identified by the Catalytic Properties of the Enzyme-Antibody Precipitates Obtained in Gel Diffusion Media  

Acetyl-D, L-phenylalanine-2-naphthyl ester Acetyl-L-tyrosine ethyl ester D-Fructose diphosphate H2CO3 

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Another important procedure is to screen for production of the desired protein by using immunological methods with antibodies raised against it. 

Studies using radioactivity-labeled acrylonitrile indicate that acrylonitrile or its metabolites form covalent adducts with cellular macromolecules in most tissues. Studies to develop chemical or immunological methods for measuring these adducts would be especially valuable in detecting and perhaps even quantifying human exposure to acrylonitrile. Adverse health effects demonstrated following exposure to acrylonitrile, particularly acute exposures, were characteristic of cyanide toxicity. Because these effects are also indicative of exposure to many other toxicants, additional methods are needed for more specific biomarkers of effects of acrylonitrile exposure. 

Craig, O.E. and Collins, M.J. (2000). An improved method for the immunological detection of mineral bound protein using hydrofluoric acid and direct capture. Journal of Immunological Methods 236 89-97. 

Substances when applied to human skin might exert a sensitizing potential on the skin and need, therefore, to be evaluated and classified for their possible toxicity. Every substance that provokes immunologically mediated cutaneous reactions (i.e., skin sensitization or allergic contact dermatitis) is referred to as skin sensitizer. Several tests are recommended, but no single method is able to identify all potential substances capable of inducing sensitization of human skin. Widely used test methods for the investigation of skin sensitization, the so-called adjuvant and nonadjuvant tests, are described below. 

It is to be expected that these extremely widely used oral methods of contraception will eventually be replaced by luteinizing hormone analogs as antiovulatory agents, or possibly by immunological methods directed against human chorionic gonadotrophin (hCG), which has both LH and FSH activity. Prostaglandins also show promise, and reliable male contraception is also a future possibility. 

An overview of the analytical techniques most frequently used that provide molecular and crystalline structure is illustrated in Scheme 1.8. Basically, they can be grouped into histochemical and immunological methods, diffraction, spectroscopic, spectrometric, chromatographic, and thermoanalytical techniques. 

Western blotting has become an important, modern technique for analysis and characterization of proteins. The procedure consists of, first, the electrophoretic transfer (blotting) of proteins from polyacrylamide gels to synthetic membranes. The transferred blots are then probed using immunological detection methods to identify proteins of specific structure and/or function. In this experiment, bovine serum will be fractionated by SDS-PAGE and the proteins blotted onto a nitrocellulose membrane. Serum glycoproteins will be identified by their specific interaction with the lectin concanavalin A. 

Although microbiological and HPLC methods are the most commonly used methods for analysis of vitamins, there are some immunological methods... 

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