Enzyme donor

Fig. 2.U The variation of logA , with of the buffer for reaction (2.166). The buffers used are Mes(l), 3,5-lutidine(2), 3,4-lutidine(3), 2,4-lutidine(4), 1-Meimid(5), Hepes(6), triethanolamine(7), 4-Meimid(8), l,2-diMeimid(9), Ted(lO) and Ches(ll). The curve drawn is calculated for = 1.1 x 10 M s and a pA n for the enzyme donor group of 7.6. Reprinted with permission from R. S. Rowlett, and D. N. Silverman, J. Amer. Chem. Soc. 104, 6737 (1982). (1982) American Chemical Society.

HOIA using cloned enzyme donor immunoassay (CEDIA )... 

This technique uses two different inactive enzyme fragments, a large fragment called enzyme acceptor (EA) and a very small fragment called enzyme donor (ED). Those fragments can associate to give an active enzyme. 

Both competitive and noncompetitive methods have been incorporated into homogeneous enzyme-labeled immunoassay kits that ultimately relate enzyme activity to analyte concentration.22 The competitive-binding assays are called enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescein immunoassay (SLFIA), apoenzyme reactivation immunoassay (ARIS), and cloned enzyme donor immunoassay (CEDIA), while a noncompetitive method is called enzyme inhibitory homogeneous immunoassay (EIHIA). 

Cloned Enzyme Donor Immunoassay. This technique, abbreviated CEDIA, has arisen as a result of recombinant DNA technology, and a fundamental understanding of the enzyme p-galactosidase. This enzyme is active only in its native tetrameric structure the four identical subunits comprising the tetramer are inactive if the quaternary structure of the holoenzyme is absent. It is now known that if any of the amino acids between positions 10 and 60 from the N-terminal end of the p-galactosidase subunit are absent, an active tetramer cannot be formed. 

Given this information, researchers have devised donor and acceptor components of the p-galactosidase subunit. The enzyme donor is conjugated to antigen, and becomes the indirect label in a competitive immunoassay. If this labeled antigen is not bound to antibody, then the combination of donor and acceptor result in a complete subunit that can combine with three other complete subunits to form an active tetramer. If the labeled antigen is antibody-bound, steric effects prevent donor-acceptor combination, so that the generation of active enzyme is not possible from this potential subunit. The concepts in this assay are shown in Eq. 6.18 ... 

Figure 9-15 CEDIA and EMIT homogeneous immunoassays. A, Enzyme acceptor ED, enzyme donor.

The cloned enzyme donor immunoassay (CEDIA) technique for T4 measurement is based on the use of inactive fragments of p-galactosidase called enzyme acceptor (EA) and enzyme donor (ED). In this method, T4 in a serum sample competes with ED-labeled T4 for a hmited number of anti-T., binding sites. As the concentration of unlabeled T4 increases, less ED-labeled T4 binds to the antibody and more reassociates with EA to form an active enzyme. The catalytic activity of the holoenzyme increases in proportion to the amount of T4 in the sample. This method has been adapted for use on on routine chemistry analyzers. ... 

The [3-galactosidase analog consists of two subunits a large dimeric polypeptide (200 kDa), denoted enzyme acceptor (EA)2, and a small polypeptide (20 kDa), denoted enzyme donor (ED). (EA)2 and ED are enzymatically inactive but spontaneously associate to give enzymatically active tetramers. In CEDIA assays, [45,46] the hapten or analyte is covalently linked to the ED, and an analyte-specific antibody is used to inhibit the assembly of the enzymatically active tetramers. Analyte in a patient s serum competes with the analyte in the analyte-ED conjugate for antibody, modulating the amount of active B-... 

I van der Weide, et al. Evaluation of the cloned enzyme donor immunoassay for measurement of phenytoin and phenobarbital in serum. Ther Drug Monit 15 344, 1993. 

All immunoassays are based on the principle detection method employed. Commonly used meth-of competitive displacement of a labeled drug ods include fluorescence polarization, enzyme im-from an antigen-antibody complex by unlabeled munoassay, cloned enzyme donor immunoassay, drug in the sample. The fundamental difference enzyme-linked immunosorbent assay, and radioin the currently available immunoassays is the immunoassay. 

Enzyme Donor/glycoside Acceptor/nucleophile Product(s) ... 

Cloned enzyme donor immunoassays, 1035 Cloverleaf structure, 2703 Cloves, 3664, 3666, 3671, 3673 CLP. See Caulerpin (CLP)... 

---