Immunochemical complex

Much research has gone into raising the sensitivity and selectivity of immunosensors to the desired levels. Several labels have proved to ensure a high sensitivity, yet radioisotopic labels have essentially been avoided. Non-isotopic labels for immunosensors include various enzymes, catalysts, fluorophores, electrochemically active molecules and liposomes. Labelled immunosensors are basically designed so that immunochemical complexation takes place on the surface of the sensor matrix. There are several variants of the procedure used to form an immunocomplex on the matrix. In the final step, however, the label should always be incorporated into the immunocomplex for determination, as shown in Fig. 3.27.B. 

Some electrochemically active substances that can generate photons on an electrode surface are suitable labels for homogeneous immunoassays. A labelled antigen exhibits an electrochemical reactivity and produces luminescence, but when it is immunochemically complexed, the labelled antigen loses its electrochemiluminescent properties. One optical immunosensor for homogeneous immunoassays was assembled by spattering platinum on the end surface of an optical fibre. Spattered platinum maintains optical transparency and functions as an electrode. An optical electrode efficiently... 

Antibodies (ab) are high-molecular weight soluble proteins (immunoglobulins) produced by organisms in response to foreign substances (antigens, ag), with whom they form immunochemical complexes ... 

Direct immunosensors register immunochemical complex formation at the transducer surface via electrochemical, mass or optical changes. The advantage of a direct immuno-sensor is that measurement of the antigen-an-... 

Common Procedures. The general analytical scheme for immunochemical methods is rather simple. The analyte of interest, the antigen (Ag), reacts with the analytical reagent, the corresponding antibody (Ah), forming an immunochemical antigen—antibody complex ... 

Va.ria.tions in Methods. The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be cmde antisemm, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is mn, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

An electrode in which an antibody or an antigen/hapten is incorporated in the sensing element is termed an immunoelectrode . The potential response of the immuno-electrode is based on an immunochemical reaction between the sensing element of the electrode and antibody or antigen/hapten in the sample solution. One example of such an electrode is the polymer membrane electrode shown in Fig. 7. The selective response of this electrode to specific immunoglobulins is based on the interaction between antibody in solution and an antigen-ionophore complex in the membrane ... 

The application of PSA measurements for clinical monitoring of prostatic carcinoma requires fine tuning of PSA assays. One important aspect of this tuning is to have well-defined standards (primary calibrators). Calibrators or primary reference materials consisting of PSA complexed with ACT have been prepared and are available to sponsors of commercial immunoassays. As a result of this, some sponsors have studied calibration stability and have shown that calibration did not change within 14 to 90 days. Primary references of 90 percent PSA-ACT and 10 percent f-PS A have been shown to minimize differences in PSA measurements between different assays [NCCLS Document—Primary Reference Preparations Used to Standardize Calibration of Immunochemical Assays for Serum Prostate... 

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. 

Because of the complex and polymorphic nature of the Lp(a) lipoprotein, together with the homology of the apo(a) moiety with plasminogen, a number of specific problems arise concerning the immunochemical quantification of Lp(a). These include the selection of a suitable type of immunoassay, its specificity and sensitivity, and the type of antisera used in the assay (L2). Moreover, the selection of an appropriate standard and of the units of mass to express the amount of Lp(a) require careful consideration (L4). 

Multiple forms of cytochrome P-1+50. It is now clear that there are more than one form of cytochrome P-1+50. Thomas et al. have recently shown by immunochemical means that there are at least six forms of mammalian cytochrome P-1+50 (39) In 1960 s it was noted that there are at least two catalytically and spectrally distinct cytochrome P-l+50 s, viz. cytochrome P-1+50 and cytochrome P-1+1+8 or P -1+50 (1 0, 1 1). Cytochrome P-1+1+8 is inducible by PAH s such as 3-methylcholanthrene (MC) and BP. It metabolizes preferentially PAH s (such as the above carcinogenic inducers). Cytochrome P-1+1+8 derives its name from the fact that when reduced and complexed with carbon monoxide it has an absorbance maximum at 1+1+8 nm. Cytochrome P-1+50 induced by compounds such as phenobarbital (PB) appears similar to the control cytochrome P-1+50 both spectrally and catalytically. 

The invention of B cell hybridoma technology (K3) has allowed the generation of various kinds of useful antibodies, even very minor or rare antibody species elicited in serum by the conventional procedure, as a pure immunochemical reagent in almost unlimited amounts. Among such new-generation monoclonal antibodies, anti-idiotype antibodies and anti-immune complex (anti-metatype) antibodies have been successfully introduced as key reagents enabling noncompetitive hapten immunoassays (Fig. 11). 

Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. It involves the interaction between an antigen and its specific antibody. Antigen-antibody interactions may produce a network of many antigen molecules cross-linked by antibody molecules, which result in insolubilization and precipitation of the complex (Williams 2000). 

Chapters 17 through 21 deal with carbohydrate-enzyme systems. Hehre presents some new ideas on the action of amylases. Kabat presents some new immunochemical studies on the carbohydrate moiety of certain water-soluble blood-group substances and their precursor antigens. Hassid reviews the role of sugar phosphates in the biosynthesis of complex saccharides. Pazur and co-workers present information obtained by isotopic techniques on the nature of enzyme-substrate complexes in the hydrolysis of polysaccharides. Gabriel presents a common mechanism for the production of 6-deoxyhexoses. An intermediate nucleoside-5 -(6-deoxyhexose-4-ulose pyrophosphate) is formed in each of the syntheses. 

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... 

---