Immunochemical methods antibody development

Immunochemical methods have been developed and placed on the market to analyze tetracycline residues (see Table 4). Thus, a qualitative EIA has been developed and used to analyze tetracyclines in honey samples with a detection level of 20 pg/kg-1 [96]. A microplate-based indirect ELISA has been developed to analyze tetracyclines using polyclonal antibodies. The assay could measure tetracycline in the range between 0.1 and 6 ng mL L Other tetracycline antibiotics such as chlortetracycline, rolitetracycline, or minocycline are also highly recognized in this assay [98]. Several immunoassay kits are commercially available for the analysis of tetracyclines although, to our knowledge, none of them... 

Although both polyclonal and monoclonal antibodies have been effectively used in immunochemical assays, only the latter can provide the high specificity required in some applications. Antibody specificity, on the other hand, is both a major advantage and disadvantage for immunochemical methods. It allows for highly selective detection of analytes but at the same time may complicate the development of multiresidue methods. Moreover, production of monoclonal antibodies requires special expertise and it is much more expensive than polyclonal antibodies. Thus, in cases where a range of analytes similar in molecular structure are required to be determined, a polyclonal may be more suitable than a monoclonal antibody. 

Once antibodies and antigens have been developed, they must be incorporated into an assay system for visualization of the primary antibody-antigen reaction. To accomplish this task effectively, an assay format and procedures to visualize the antibody-antigen reaction must be first selected, the necessary reagents must then be prepared, and the final immunochemical method including sample preparation must be optimized for the intended application. 

The development of immunoassays for the detection of food components and contaminants has progressed rapidly in the last few years [7]. Antibodies against almost all the important food residues compounds are currently available. Classical immunochemical methods such as immunodiffusion and agglutination methods for food analyses generally involve no labeled antigen or antibody. Concentration of the antigen-antibody complex is estimated from the secondary reaction that leads to precipitation or agglutination. These methods are not sensitive, are subject to... 

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... 

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. 

One of the most important contributions of immunochemistry to daily practice is the development of immunoanalysis. The interest in immunochemical methods increased when sensitive labels for detecting the complex antigen-antibody were introduced. The labeling of antibodies or antigens with radionuclides increased the sensitivity of immunochemical methods so remarkably that the determination of specific compounds at ng levels is quite common and under certain circumstances even much lower concentrations may be detected. 

Selective antibodies are the basis of all immunochemical methods and therefore the development of these antibodies is fundamental to this technology. In the early days of immunochemistry, only polyclonal antisera had been developed, and they have been used for more than 25 years both in academia and in commercial test-kits. Although the sensitivity and selectivity of polyclonal antisera are excellent, these reagents are a mixture of antibodies, and even with the same immunogen different animals will produce... 

Immunochemical methods are very versatile, and a range of antibodies for small environmentally relevant compounds has been developed over many years, but... 

Analytical Methods for Blood. Methods to measure sulfur mustard adducts to DNA in white blood cells have been developed using LC with fluorescence detection (Ludlum et al., 1994) and using an enzyme-linked immunosorbent assay (ELISA) (van der Schans et al., 1994, 2004). The DNA adduct that appears to be the most abundant results from sulfur mustard attachment to the N7 position of deoxyguaninosine (Fidder et al., 1994). The immunochemical method developed by van der Schans used monoclonal antibodies that were raised against N7-(2-hydroxyethylthioethyl)-guanosine-5 -phosphate. 

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. 

Our approach is based upon the development of monoclonal antibodies against adducts of sulfur mustard with DNA and proteins for use in a variety of immunochemical assays, and upon the development of procedures for GC-MS and LC-MS-MS analyses which can be used to validate the immunochemical assays. Within this framework, an immunochemical assay was developed with monoclonal antibodies raised against the N7-adduct of sulfur mustard with T-deoxyguanosine in DNA (N7-hydroxyethylthioethyl-2 -deoxyguanosine N7-HETE-dG see Figure 1), enabling the sensitive detection of this adduct in DNA of human white blood cells and in DNA of human skin after exposure to sulfur mustard (2). Using this method, the presence of N7-HETE-dG in blood samples taken from Iranian victims from the Iran - Iraq war could be assessed (3). 

Sulfur mustard adducts were clearly detected in the stratum corneum whereas DNA counterstaining visualizes the presence of DNA in the nucleated cells. Hardly any fluorescence due to antibody treatment was measured over the nonexposed skin cross-section at the conditions used. In contrast to the immunochemical method for analysis of DNA-sulfiir mustard adducts, which involves laborious workup procedures, this approach opens the way for development of a rapid detection kit that can be applied directly to the skin. 

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