Quantitative analysis immunochemical

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. 

Oudin, J., Immunochemical analysis of human serum and its fractions. II. QuaUta-tive and quantitative analysis of the fraction soluble in two-thirds satiuated ammonium sulphate. J. Immunol. 81, 373-388 (1958). 

Clausen R, Weller M, Wiedemann P, Heimann K, Hilgers RD, Zilles K. An immunochemical quantitative analysis of the protein pattern in physiologic and pathologic vitreous. Graefes Arch Clin Exp Ophthalmol 1991 229 186-190. 

Hamana, K., N. Iwasaki, T. Takeya, and H. Takita. 1996. A quantitative analysis of rat central nervous system myelination using the immunochemical method for MBP. Brain Research Developmental Brain Research 93 18-22. 

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. 

Because of the simplicity of the immunochemical procedure based on the biocomposite affinity platform, this strategy can be suitable for fast semiquantitative and quantitative on-site analysis for the presence of atrazine (or atrazine immunoreactive herbicides) in real samples. The fabrication of the biocomposite-based biosensor can be easily transferred... 

Several qualitative and quantitative immunochemical methods and their application to the analysis of environmental samples have been described for OP insecticides, a family that includes widely used pesticides such as azinphos-ethyl/methyl, dichlorvos, fenitrothion or fenthion, malathion, mevinphos, and parathion. Mercader and Montoya202 produced monoclonal antibodies against azinphos-methyl and developed an ELISA that was used for the analysis of water samples from different sources, reaching detectability levels near 0.05 pg I. Watanabe et al.203 reported the production of polyclonal antibodies and ELISA procedures to analyze fenitrothion in river, tap, and mineral water (LOD = 0.3 pg L ). Banks et al.204 produced polyclonal antibodies against dichlorvos, an organophosphate insecticide used for stored grain, which also cross-reacts with fenitrothion. Nishi et al.205 reported the first immunoassay for malathion. Residues of this insecticide have... 

In 1970, Eckman et al. devised the automation of a quantitative immunochemical analysis of transferrin (El). In this automated flow system, diluted samples were allowed to react with antitransferrin antiserum serially and the degree of light scattering of the resulting turbidity was measured in the fluorometer, used as a nephelometer. The optimal conditions for nephelometry were extensively studied. Subsequently, Buffone reported... 

Assays for biological activity, where applicable, should be part of the pivotal stability studies. Appropriate physicochemical, biochemical and immunochemical methods for the analysis of the molecular entity and the quantitative detection of degradation products should also be part of the stability program whenever purity and molecular characteristics of the product permit use of these methodologies. 

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. 

Quantitative fluorimetric analysis on real samples usually requires a separation of the fluorescent analyte from its matrix because of the wide occurrence of fluorescent and quenching impurities. Occasionally, simple solvent extraction will do the trick. More often, chromatographic separation is necessary, and the fluo-rimeter becomes merely a detector for the chromatographic eluates. Recently, however, the application of immunochemistry to analytical chemistry has permitted in-situ analysis on a scale never before possible. Although the earliest immunochemical analyses utilized radioisotopes, the most popular ones used today employ fluorescent probes or labels. 

Fichtinger-Schepman, A. M. J. Baan, R. A. Luiten-Schuite, A. Van Dijk, M. Lohman, P. H. M. Immunochemical quantitation of adducts induced in DNA by cis diamminedichloroplatinum-II and analysis of adduct-related DNA-unwinding. Chem-Biol. Interact., 55 275-88. 1985. 

In general, immunochemical analyses of antibody-antigen interactions can provide both qualitative and quantitative information. Howevo-, these approaches lack the ability to provide information regarding the detailed locations of epitopes on antigens. In order to obtain such information, one must conduct structural analysis on antibody-antigen complexes. 

Immunoreagents are well suited for quantitation of amplified DNA. However, the exponential nature of PCR amplification makes it difficult to extrapolate from the amount of amplified DNA to the amount of starting material. The most reliable quantitative PCR methods involve real-time assay of amplified DNA during the exponential phase of the amplification reaction. Because the analyte of interest is usually the DNA template, not the amplification product, immunochemical methods at their present stage of development are generally best applied to an unamplified template where concentrations permit direct analysis or for qualitative assays. 

J.M. Wilson, P.E. Daddona, H.A. Simmonds, K.J. Van Acker, and W.N. Kelley, Human adenine phosphoribosyltransferase immunochemical quantitation and protein blot analysis of mutant forms of the enzyme, Biol. Chem. 257 1508-1515 (1982). 

Immunochemical analysis is a convenient approach for environmental analysis provided appropriate antibodies and format have been developed. Often, the assay depends on a colorimetric determination for its quantitative result. The advantage of immunochemical analysis is its specificity as well as its speed. Specific references should be consulted on applications. 

---