Labeled immunochemical assays

A number of immunochemical techniques have been used to quantify analytes of clinical interest. They include radial diffusion (RID) and electroimmunoassays, turbidimetric and nephelometric assays, and labeled immunochemical assays. 

Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

Immunochemical assay procedures may be divided into two classes, direct and indirect. Direct methods involve synthesis or modification of the molecule to be measured in the presence of a radioactively labeled precursor followed by its isolation using immunochemical precipitation. Indirect methods involve competition of a nonradioactive protein obtained from an experimental sample with a radioactively labeled standard protein for a limited amount of antibody the quantity of desired protein... 

Howanitz, J. H. (1992). Overview of Nonisotopic Immunoassay Labels in Immunochemical Assays and Biosensor Technology for the 1990s, Yasushi Kasahara Robert M. Nakamura, Garry A. Rechnitz (Ed.), Amer Society for Microbiology, pp. 22-35. 

The previously discussed methods rely on evaluating the immune complex formation as an index of antigen antibody reaction. As demonstrated previously in equation (1), the overall reaction occurs in sequential phases, and only the final phase is the formation of the immune complex. However, the initial binding of the antibody and antigen has also been demonstrated to be very useful analytically and has been used with labeled antigens and antibodies to develop many sensitive and specific immunochemical assays. The reaction describing this initial binding and the kinetic constant for the overall reaction are shown in equations (4a) and (4b), respectively ... 

Competitive Immunoassays. In a competitive immunochemical assay, all reactants are mixed together either simultaneously or sequentially. In the simultaneous approach, the labeled antigen (Ag ) and unlabeled antigen (Ag) compete for binding to the antibody. In such a system, the avidity of the antibody for both the labeled and unlabeled antigen must be the same. Under these conditions, the probability of... 

Immunochemical assays that require a separation of the free from the bound label are termed heterogeneous those that do not are called homogeneous. 

In the decade following the pioneering developments of Yalow and Berson, all immunoassays used a radioactive label in a competitive assay format. Since the introduction of enzyme immunochemical assays in the 1970s, a vast array of sophisticated immunochemical assays have evolved and have been very widely applied. Specific examples of several of these assays follow others are briefly described in Box 9-4. 

The immunochemical interaction between the antigen and antibody is very specific. By labeling either the antigen or antibody, the method s sensitivity is increased. The most frequently used labels to increase sensitivity are radionucHdes (see Radioisotopes) where the assay process is called radioimmunoassay (RIA), or en2ymes where the assay is named en2yme immunoassay (ElA) (see Enzyme applications). 

V) The validity of RIA entirely depends upon the identical behaviour of standard and labelled substance unknown, and not on the identity of the labelled tracer and the unknown. Hence, the experimental conditions of incubation of standards and unknowns must be identical for any factors that might affect the extent of the immunochemical reaction, pH, ionic composition, protein content or any other substances of interest. However, these conditions may be tested conveniently and can be controlled effectively by preparing standards in hormone free plasma at the same dilution at which unknowns are assayed. 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

The use of rare-earth elements as fluorescent labels, e.g. europium seems to be very promising. Their application in immunochemical techniques permits assays of high sensitivity which are not troubled by non-specific short-lived fluorescence since this can be eliminated by time-resolution fluorometers... 

Apart from assays of proteolytic activity and immunochemical methods (7.1 and 7.5), protein labeling is exploited in other areas of analysis. Noteworthy among them are protein determination in complex mixtures or even directly in cells, protein separation (affinity chromatography and electrophoresis) and protein detection following various separation techniques. For this purpose different labels are applied. 

The specific activity of proteins assayed by direct immunochemical methods or those used as standards in radioimmunoassays profoundly affects the resolution attainable by these techniques. Therefore, the nature of the radioactive label to be used in such experiments must be considered carefully. Table 8-6 lists the isotopes available for this purpose along with the number of atoms of each isotope that must be incorporated to produce an arbitrary counting rate. As can be seen here, 557 atoms of H and 261,672 atoms of C must be incorporated into every molecule of protein to yield the same number of disintegrations per minute as only one I or 11 S molecules. S-methionine is often the isotope of choice for many direct immunochemical procedures since it is relatively inexpensive to prepare at high specific activity. On the other hand, the relative ease with which radioactive iodine may be incorporated into a purified antigen makes it the isotope of choice for radioimmunoassay methods. Of the two iodine isotopes available, is most often used because of its longer half-life. This is an important consideration since it usually takes more than 1 week to prepare and test a labeled antigen prior to its experimental use. 

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