Immunochemical techniques

In response to exposure to an antigen, microorganisms and other living entities produce antibodies. Immunological techniques rely on the interaction between an antigen and an antibody specific for that antigen. The antigen may be a microbial cell wall or capsule, a characteristic cellular component, or even a specific metabolite. 

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

Gene function in cultured cells can be investigated by expression of the gene product in a suitable expression system or, in vivo, by the creation of transgenic mice, either knockout mice in which the gene in question has been functionally deleted or mice into which a transgene has been introduced. 

A general, but more detailed and specific, account of these methods may be found in Smart (2001 see Suggested Reading). 

Most of the recently developed methods for the detection, characterization, and quantitation of proteins are immunoassays based on the fact that proteins are antigens, compounds that can be recognized by an antibody. It is also true that by combining small molecules (haptens) with a larger carrier molecule such as a protein, these methods can be extended to small molecules of interest since antibodies can be produced that recognize epitopes (specific sites on the antigen recognized by the antibody) that include the hapten. 

Immunolocalization is a technique for identifying the presence of a protein within the cell, its relative abundance and its subcellular localization. After suitable preparation of the cells, they are treated with an antibody (the primary antibody) that binds to the protein of interest. An antibody that binds to the primary antibody (the secondary antibody) is then allowed to bind and form an antigen—primary antibody—secondary antibody complex. The detection system generally consists of the formation of a colored insoluble product of an enzymatic reaction, the enzyme, such as alkaline phosphatase or horseradish peroxidase, being covalently linked to the secondary antibody. 

Labels used in fluorescence immunoassays are mostly based on fluorescein 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

Substrate products can be classified as either soluble or precipitating. Soluble peroxidase substrates include o-phenylenediamine, which is converted into a yellow product 2,2 -azino-(3-ethyl)-benzothiazoline-sulfonic acid, which is converted into a green product and tetramethylbenzidine, which is converted into a blue product. Precipitating substrates for peroxidase include 4-chloronaphthol, which yields a blue precipitate and aminoethylcarbizole, which forms a red precipitate. Alkaline phosphatase is most frequently used with p-nitrophenyl phosphate to give a yellow-orange soluble product, or with 5-bromo-4-chloro-3-indo-lyl-phosphate p-toluidine salt to yield an insoluble blue product. 

Many types of mono-, bi-, and multifunctional coupling reagents are available for labeling antibodies or antigens with an enzyme. Glutaraldehyde, carbodiimide, N-succinimidyl-3-(2-pyridyldithio)propionate, and periodate oxidation of carbohydrate moieties to form active dialdehydes are several commonly used approaches in the preparation of enzyme conjugates (104-106). 

Materials commonly use as solid supports include polystyrene, polyvinyl, nylon, glass, nitrocellulose, silica, polyacrylamide, or polystyrene beads. Separation of the bound from the free reagents can be achieved through either filtration for particulate solid supports such as agarose, polyacrylamide, and polystyrene beads, or centrifugation. For disposable forms of solid supports such as multiwell plates, plastic tubes, cuvettes, balls, and dipsticks, separation can be performed through simple rinsing steps. 

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J. Clausen, Immunochemical Techniques for the Identification and Estimation of Macromolecules, 3rd ed., Elsevier, Amsterdam, the Netherlands, 1988. A. Kawamura, Jr., ed.. Fluorescent Mntibody Techniques and Their Applications, University of Tokyo Press, Baltimore, 1977. 

Mycotoxins, toxic metaboUtes of some fungi, can be assayed by immunochemical techniques to determine concentration in animal feed and foodstuffs. Some of the analytes assayed in kits and the detection limits are Hsted in Table 4 (45). These assays are especially advantageous for routine analysis of large samples of foodstuffs (45,46). 

The use of immunoassays for the determination of pesticides and veterinary medicines in food animals has increased since the early 1990s. The advantages of simple analysis, quick results, and high throughput make immunoassays a powerful technique for problematic matrices commonly encountered in animal agriculture. Careful development and validation are required to obtain accurate results, however. This review has demonstrated that most immunochemical techniques have been designed for use with milk samples, but a number of applications have also been developed for liver and muscle samples. The development of immunoassay techniques for residue analysis in eggs has clearly not been pursued to the extent of other edible tissues. 

Volume 84. Immunochemical Techniques (Part D Selected Immunoassays) Edited fey John J. Langone and Helen Van Vunakis... 

Volume 92. Immunochemical Techniques (Part E Monoclonal Antibodies and General Immunoassay Methods)... 

Volume 108. Immunochemical Techniques (Part G Separation and Characterization of Lymphoid Cells)... 

Volume 121. Immunochemical Techniques (Part I Hybridoma Technology and... 

Volume 150. Immunochemical Techniques (Part K In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors)... 

Clausen, J. (1988) Immunochemical techniques for the identification and estimation of macromolecules. In Laboratory Techniques in Biochemistry and Molecular Biology (R.H. Burdon, and P.FL Knippenberg, eds.), 3rd edn., Vol. 1, Part 3. Elsevier, New York. 

Nithipatikom, K., and McGown, L.B. (1987) Homogeneous immunochemical technique for determination of human lactoferrin using excitation tranfer and phase-resolved fluorometry. Anal. Chem. 59, 423. 

Immunoelectron microscopy is not limited to nucleic acid localization but is also an essential component in the localization of a specific protein, polysaccharide, or theoretically any hapten under study. Therefore, immunoelectron microscopy is a valuable tool when it comes to the study of gene expression. Electron microscopy is a valuable tool in molecular biology and is even more powerful when combined with immunochemical techniques. 

Keywords Immunochemical techniques Surfactants Polyhalogenated compounds Bisphenol A Phthalate esters... 

Immunochemical techniques are based on the immunological reaction derived from the binding of the antibody to the corresponding antigen. This reaction is reversible and is stabilized by electrostatic forces, hydrogen bonds, and Van der Waals interactions. The formed complex has an affinity constant (k j that can achieve values around the order of 1010 M. This great affinity and specificity between the specific antibody and the antigen (or the analyte) have turned these techniques into powerful analytical tools to detect and quantify... 

Several immunochemical techniques have been developed as analytical tools or in sample treatment methods to separate an analyte from complex matrices. Some of the most important or more frequently used are described below. 

In recent years many efforts have been made to develop immunochemical techniques integrating the recognition elements and the detection components, in order to obtain small devices with the ability to carry out direct, selective, and continuous measurements of one or several analytes present in the sample. In this context biosensors can fulfill these requirements. Biosensors are analytical devices consisting of a biological component (enzyme, receptor, DNA, cell, Ab, etc.) in intimate contact with a physical transducer that converts the biorecognition process into a measurable signal (electrical or optical) (see Fig. 4). In... 

Fig. 6 General structures of the most important surfactants and metabolites alkylphenol polyethoxylate (APE) alkylphenol (AP) alkyl ether (AE) alkylphenol ethoxy carboxylate (APEC) linear alkylbenzenesulfonates (LAS) alkyltrimethylammonium compounds (ATMAC) dialkyldimethylammonium compounds (DADMAC) alkyldimethylbenzylammonium compounds (ADMBAC) esterquat (EQ) diesterquats (DEQ). X is usually a chlorine or bromine atom. DDAC (didecyldimethylammonium chloride) and BDD12AC (benzyldimethyldode-cylammonium) are the two target analytes with a reported immunochemical technique developed for their analysis [153,154]...

Table 4 Immunochemical techniques developed for the detection of surfactants. The sensitivity of the method and the matrix considered are shown... 

Table 5 Immunochemical techniques developed for the detection of organochlorinated substances... 

Several attempts have been made to set up immunochemical techniques for dioxin analysis (reviewed in [230,238,239]). Frequently the detectability and selectivity accomplished have not been considered appropriate for the direct analysis of environmental samples. We should notice that due to the poor solubility of PCDDs and PCDFs in water, the levels of these contaminants in aqueous samples is very low. For this reason analysts usually prefer the use of chromatographic and spectrometric methods that perform using organic solvents. However, the speed and high sample throughput that can be accomplished with the immunochemical methods have prompted several research groups and companies to establish immunochemical methods. 

It has been widely demonstrated that immunochemical techniques offer a good alternative to conventional methodologies in many areas due to the high sensitivity and selectivity achieved for the antibodies toward the target analytes. 

Keywords Immunochemical techniques Antibiotics Steroid hormones Analgesics Cytostatic agents... 

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