Immunochemical techniques quantitative methods

Coupling chromatographic procedures with immunochemical techniques can also provide a very sensitive and specific analytical system for either determinative or confirmatory analysis. If the antibody used is very specific for the analyte of interest and the antibody reactivity is known to be sensitive to small variations in the structure of the analyte tested, positive reactions with the method are strongly indicative that an analyte of defined structural characteristics is present in the sample. Full rigorous confirmation, however, would depend on further analysis by mass spectrometry, which is the method of choice in confirmatory analysis. Mass spectrometry gives specific information on the identity and structure of the compound of interest. Coupled with chromatographic techniques it becomes a very powerful confirmatory tool for both quantitative and qualitative assessment of drug residues in foods. 

During in vivo studies under biologically relevant conditions, the cis-Pt loading of the DNA is much lower than for the above-mentioned in vitro studies. It has been calculated that mortality of HeLa cells occurs at an value of 10 5 (i.e., one bound cis-Pt molecule per 105 nucleotides) (64a). This excludes atomic absorption spectroscopy for identification of the in vivo adducts. Immunochemical techniques, however, have shown to be very promising, and high sensitivity and selectivity levels have been reached. At the moment, only a few studies in which antibodies are raised against cis-Pt-treated DNA (64) or against synthetic cis-Pt adducts with mono- or dinucleotides are available (64a). With the latter method, quantitation of the different platinum-DNA adducts formed under in vivo conditions is possible. At the moment, femtomole (10-15 mol) amounts of the adducts can be detected with competitive enzyme-linked immunosorbent assay (ELISA) techniques. It has been demonstrated in this manner that the GG-Pt adduct is also the predominant adduct under in vivo conditions. 

Immunochemical methods are rapidly gaining acceptance as analytical techniques for pesticide residue analysis. Unlike most quantitative methods for measuring pesticides, they are simple, rapid, precise, cost effective, and adaptable to laboratory or field situations. The technique centers around the development of an antibody for the pesticide or environmental contaminant of interest. The work hinges on the synthesis of a hapten which contains the functional groups necessary for recognition by the antibody. Once this aspect is complete, immunochemical detection methods may take many forms. The enzyme-linked immunosorbent assay (ELISA) is one form that has been found useful in residue applications. This technique will be illustrated by examples from this laboratory, particularly molinate, a thiocarbamate herbicide used in rice culture. Immunoassay development will be traced from hapten synthesis to validation and field testing of the final assay. 

The quantitative precipitin method presupposes availability of fairly pure antigens and the successful production of specific antisera free of antibodies to contaminant proteins. Immunochemical techniques are available to ascertain whether or not these criteria have been fulfilled. Nevertheless, the possibility of cross-reactions of specific antibodies with other closely related antigens must be considered a limitation of the method. 

The sensitivity and specificity of the quantitative precipitin method make it applicable to the identification and estimation of abnormal plasma proteins in certain diseases, particularly when present in concentrations too low to be detected by other methods. Goettsch and her associates (103, 104) and Kendall (181) employed immunochemical techniques in the study of nephrotic sera Kendall (181) also investigated the distribution of his immunologically distinct globulin subfractions in hyperglobulinemia due to lymphogranuloma venereum, cirrhosis of the liver, and rheumatoid arthritis Kabat (261) was able to demonstrate the presence of as little as 0.15 gram per cent Bence-Jones protein in the serum of a patient with multiple myeloma. Many other applications of immunochemical techniques are noted by Kabat (167) and Treffers (358) in recent reviews. 

Typically the two most commonly encountered gel-based methods for quantitative immunochemical studies are RID umnunoassay and electro immunoassay ( rocket technique). 

Alkylpurines derived from DNA adducts may also be detected by HPLC equipped with fluorescence or electrochemical detection, and immunochemical methods. Mass spectrometry techniques are frequently used to obtain structural confirmation of the identity of a urinary product quantitated using GC-nitro-samine-selective detection [47], GC FID [48,49], or immunoassays [50,51],... 

Immunochemical methods are highly sensitive techniques for the detection and identification of protein targets by antigen-antibody specific reactions. Radial immunodilfusion and western blotting are among the most used techniques for the quantitative estimation of processed food and bioproduct proteins based on their immunoreactivity with specific antibodies. 

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