Definitions And Concepts

Immunoassays are applied for the measurement of both single and multiple analytes [1]. Chemical pesticides, polychlorinated biphenyls (PCBs) and antibiotics are typical examples of contaminants, which can be rapidly and efficiently determined by antibody-based methods. Antibodies enable not only a rapid detection of analyte in water, body fluids, soil or food extracts, but they can also be exploited in sample preparation prior to analyte detection. Because antibody affinity and specificity determine primarily the analytical capability of the immunochemical method, the properties of the antibodies represent an important innovative factor in developing an analytical system. Highly sensitive detection of toxic analytes can be performed by enzyme immunoassays, immunosensors and related techniques, whereas immunoaffinity chromatogra-phy and flow-injection immunoassay (FIIA) systems enable the concentration and clean up of the analytes in question.

The manner of chemical binding of the hapten to a protein determines the character of the antibody specificity. Various types of hapten derivatives conjugated to proteins have been used for antibody development. Different types of Abs are involved in immunoassays: polyclonal, monoclonal and recombinant. Polyclonal Abs are obtained from the serum of animals immunized with a particular Ag. The Ab pool obtained from serum is the result of many B-cell clones, each secreting one specific Ab. Antiserum refers to a pool of serum containing all of the Ab fraction plus other serum proteins. Whereas, monoclonal antibodies are produced following the fusion of myeloma cells with Ab-secreting B-cells (Figure 2). The resultant continuous cell line (hybridoma) produces large quantities of the homogeneous, well-defined, single-epitope Ab. The availability of large quantities of continuously produced Ab allows greater standardization and quality control of the Ab reagent. Therefore, monoclonal antibodies are more precisely characterized, legally protected and have greater acceptance by regulatory agencies.

Preparation of recombinant antibody fragments with novel binding properties was a primary goal of gene technologies. Their major asset lies in the possibility to focus mutagenesis on that part of gene that determines the structure and affinity of antibody binding site. Large phage libraries expressing antibody fragments on the surface of individual phage particles were used for preparation of recombinant antibodies. The systems enable separation of individual phage

Hybridoma

Antibody-producing hybridoma cloned

Hybridoma

Hybridoma screening for antibody

Monoclonal antibodies isolated for cultivation

Figure 2 Monoclonal antibodies production scheme.

particles and subsequent selection of phage antibodies from a large number of expressed phage particles.

Immunoassays offer a number of advantages in food contaminants analysis over the conventional methods, such as liquid- or gas chromatography-based methods, because immunoassays can provide fast, simple and cost effective detection, with sensitivity in most cases comparable or better to conventional techniques. In addition, the possible automation and the versatility in the applications of immunochemical techniques have also made these techniques more popular in recent years.

Main advantages of these methods are:

• Reduced time of analysis

• Low detection limits

• High throughput of samples

• Cost effectiveness for large numbers of samples

• Adaptability to field use

• Small volumes of sample and solvents are required.

Another relevant advantage is the cost effectivity of immunochemical approaches. In general, the major costs involved in food contaminants analysis are related to instruments (including maintenance), skilled personnel, solvents, reagents and time (for both sample pre-treatment and analysis). On the contrary, the instrumentation required by immunochemical approaches is economical in comparison to classical instrumental method, and involved manipulation are easy and rapid, requiring none or minimal quantities of solvent as well as little amount of sample. All these factors reverberate in more than 10 times lower cost of analysis using immunoassays than using classical instrumental analysis. On the other hand, a series of limitations should be also pointed out.

• Immunoreagent preparation

• Lack of implementation

• Lack of immunoreagent stability

• Cross-reactivity

The lengthy development time that can be required for immunoreagent preparation and the lack or limited response in front of some contaminants is sometimes an important limitation of immunochemical techniques, e.g., almost no immunological response can be obtained in front of per-fluorinated compounds and no antibodies against these contaminants can be obtained.

The lack of stability (especially thermal, and in front of extreme pH ranges) is a limitation for the use of immunochemical analysis in some cases.

Cross-reactivity is the lack of specificity of an immunoassay in front of an analyte when the immunoassay can react with other structurally related compounds, this is a characteristic that directly depends on the antibody. However, when the object of the analysis is screening a group of related substances, this fact can be an advantage.

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