Homogeneous enzyme immunoassay principle

Figure 14. Principle of homogeneous enzyme immunoassay (Reproduced with permission from Ref. 17. Copyright 1973 American Association for Clinical Chemistry, Inc.)...

Fig. 1. Schematic representation of the principle of homogeneous enzyme immunoassay developed by Rubenstein (emit). [Cited and modified from Fig. IV-4, Miyai, K., in Ishikawa et al., eds. (15).]...

Baquir et al. developed an "homogeneous" enzyme immunoassay for the determination of conjugate chenodeoxycholate. The procedure does not require extraction of serum and separation of antibody-bound from free antigen, hence, the term "homogeneous". It is not based on the same principle as the common competitive... 

EEIA may be realized in different ways. Homogeneous (non-separation) enzyme immunoassay is based on the principle of modulation of activity of the enzyme label when an antigen binds to an antibody. This means that the activity of an enzyme label may be enhanced (activation) or suppressed (inhibition). In the first case, for instance, the attachment of a thyroxine to malate dehydrogenase causes an inhibition of the enzyme. This inhibition is, however, reversed when this conjugate binds... 

The term homogeneous immunoassay may be defined as an immunoassay in which the extent of the antigen-antibody reaction can be determined without physical separation of the free and bound forms. This term is usually used for immunoassays such as enzyme and fluorescence immunoassays in which labeled substances (markers) are used. It does not include immunoassay systems such as nephelometry in which no labeled substances are used. Homogeneous immunoassays are widely used as routine tests because the procedures involved are simple. The principle of homogeneous immunoassay is based on changes in signals of the indicators by the antigen-antibody reaction (N5, U2). 

This method was developed by Collaborative Research Inc. (F3). The technique, based on the use of immunoreactive liposomes, may be classified as another type of homogeneous immunoassay. The liposomes are microscopic vesicles (200-1000 nm in diameter) consisting of a relatively impermeable lipid bilayer that delineates and separates an internal aqueous compartment from the external aqueous medium. The principle is as follows (Fig. 3 and Table 6). An enzyme, alkaline phosphatase, is encapsulated in the liposomes [E] and sequestered from the substrate, p-nitrophenyl phos-... 

Homogeneous immunoassay may serve as an example of a principal analytical approach realized by employing micellar systems [72,73] (Fig. 13). The enzyme (peroxidase) modified by hapten (thyroxine) has the same dependency profile of keat (wq) as the native enzyme (see Fig. 13). Formation of an immunocomplex (larger by size) in compliance with the principle of geometric correspondence results in a shift of the profile (wq) towards larger values of wq. At fixed values, immunocomplex formation can be denoted by a decrease in the observed reaction rate. Destruction of the immunocomplex, e.g., by introduction of free hapten, is detected by an increase in the observed enzymic activity. 

In this study two different flow-injection immunoassays are presented as well as the flexible automation system CAFCA (Computer Assisted Flow Control Analysis), which has been used for their control, uptake measurement, evaluation and visualization. Both immunoassays (a heterogeneous and a homogeneous assay) are based on the principles of flow-injection analysis and were developed for reliable, fast monitoring of relevant proteins in animal cell cultivation processes. Off-line applications of measurements of medium samples as well as online application during a mammalian cell cultivation are presented. All results are compared to results obtained with ELISA (Enzyme Linked Immunosorbend Assay). The requirements of the automation of flow-injection immunoassays with respect to their flexible control are discussed. 

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