Enzyme immunoassay, homogenous development

In another study, a microchip-based electrochemical enzyme immunoassay was developed by Chatrathi and colleagues [65] and its performance is demonstrated for the determination of monoclonal mouse IgG as a model analyte. The direct homogeneous immunoassay requires the integration of electrokinetic mixing of ALP-labeled anti-mouse IgG antibody (Ab-E) with the mouse IgG antigen (Ag) analyte in a precolumn reaction chamber, injection of immunochemical products into the separation channel, followed by rapid electrophoretic separation of enzyme-labeled free antibody and enzyme-labeled antibody-antigen complex. The separation is followed by a postcolumn reaction of enzyme tracer with p-aminophenyl... 

Homogeneous electrochemical enzyme immunoassays for both phenytoin and digoxin have been developed. In both cases the label was glucose-6-phosphate dehydrogenase, which catalyzes the reduction of NAD to NADH. The NADH produced was detected by LCEC at a carbon paste electrode. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

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).]...

The development of enzyme immunoassays was pioneered in 1972 by Engvall and Perlmann " and by Van Weemen and Schuurs and is translated into a wide variety of enzyme-based systems used both in research and in routine analysis with sensitivities approaching those of radioimmunoassays. Enzyme immunoassays (EIAs) can be divided into two major classes, homogeneous and heterogeneous. 

Ngo, T.T. Lenhoff, H.M. Enzyme modulators as tools for the development of homogeneous enzyme immunoassays. FEES Letter 1980, 116, 285-288. 

Homogeneous enzyme immunoassays have also been developed for serum T4 determination. These procedures are rapid and simple to use and have also been applied to several major automated instruments.For example, the enzyme-multiplied immunoassay technique (EMIT) for T4 measurement uses glucose-6-phosphate dehydrogenase covalently hnked to T4 as the enzyme label.Binding of T4 specific antibody to this label reduces enzyme activity, perhaps as a result of steric or allosteric inhibition As the concentration of unlabeled T4 increases, less enzyme-labeled hormone is bound by the antibody. As a result, the catalytic activity of the unbound enzyme conjugate increases in direct proportion to the amount of T4 in the specimen. The indicator reaction involves oxidation of glucose-6-phosphate with simultaneous reduction of nicotinamide-adenine dinu-... 

A homogeneous enzyme immunoassay based on antibody inhibition of enzyme conversion from the apo- to the holo- form has been developed for DNP-aminocaproic acid, DNP-ACA (Ngo et al., 1985). A competitive equilibrium is established between the analyte DNP-ACA and DNP-conjugate apoglucose oxidase, DNP-CAGO, which is the labeled hapten. Flavin adenine dinucleotide, FAD, added to the mixture binds to free DNP-CAGO to give DNP-CAGO FAD, which is enzymatically active and... 

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... 

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-... 

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is... 

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