CEDIA in vitro diagnostics with a novel homogeneous immunoassay technique. Current status and future prospects.

CEDIA assays represent a state of the art technique utilizing two genetically engineered, enzymatically inactive fragments of beta-galactosidase as the basis for a homogeneous enzyme immunoassay. The smaller, amino-terminal polypeptide, designated the enzyme donor (ED), can recombine spontaneously with the large residual fragment, called the enzyme acceptor (EA), to form active beta-galactosidase, in a process called complementation. ED have been designed in such a way that a ligand, such as a hormone or drug, can be chemically attached to a specific amino acid residue without affecting the enzyme complementation. However, the binding of a ligand-specific antibody to the ED-ligand conjugate will inhibit complementation. If a sample containing ligand is added to the reaction mixture, the ligand will compete with the ED-ligand conjugate for the limited number of antibody binding sites. Thus, the ligand concentration in the sample will modulate enzymatic activity by influencing the amount of free ED-ligand conjugate available for complementation. The basic technology of CEDIA assays has a number of inherent advantages, the most important of these being a linear calibration curve with high precision over the whole assay range, lack of endogeneous enzyme activity and minimal serum interference, chemically defined conjugates and flexibility in assay design. These provide significant advantages in comparison to other homogeneous immunoassay techniques. As a result, CEDIA assays have been successfully developed for high concentration drugs such as theophylline, phenobarbital and phenytoin as well as for very low concentration analytes such as digoxin, B12 and folate. In a modified assay format, even the determination of binding proteins has been accomplished, an example being thyroxine binding proteins in the CEDIA T-uptake assay. More recently, the methodology has been extended to the measurement of high molecular weight analytes like ferritin.

CEDIA--homogeneous immunoassays for the 1990s and beyond.

New homogeneous enzyme immunoassays have been developed for cortisol, digoxin, digitoxin, theophylline, phenytoin, and phenobarbital using the cloned enzyme donor immunoassay technology. As applied to Boehringer Mannheim/Hitachi analysis systems these methods provide rapid, accurate and precise quantification of analytes, with minimal interferences from endogenous serum constituents and low cross-reactivities to structurally-related hormonal precursors, drug metabolites and natural compounds. Additional significant features of the new assays are linear standard curves and two-point calibration. The six CEDIA assays join the two currently available CEDIA assays for determination of the thyroid parameters T4 and T Uptake. Additional new therapeutic drug and anemia monitoring assays are under development, demonstrating the versatility of the cloned enzyme donor immunoassay technology. These tests, in concert with Boehringer Mannheim/Hitachi analyzers, provide a high throughput, random access immunoassay system. The menu of available assays should continue to increase during the 1990s, providing efficient automation while allowing consolidation of testing on a limited number of instrument systems.

A new homogeneous enzyme immunoassay using recombinant enzyme fragments.

Homogeneous enzyme immunoassays have played a major role in the development of simple and easy to use diagnostic tests for clinical laboratory instrumentation. A novel homogeneous enzyme immunoassay system, CEDIA, has been developed using enzyme fragments prepared by recombinant DNA technology. Two separate genes are engineered to express two separate polypeptide fragments: enzyme-donor (ED) and enzyme-acceptor (EA). These fragments can spontaneously recombine to form active beta-galactosidase enzyme. Ligands can be attached to the ED peptide in such a way that the degree of recombination is controlled by the binding of anti-ligand antibodies to the enzyme donor-ligand conjugate. CEDIA methodology is based on the competition between ligand in the sample and ED-ligand conjugate for limiting the amount of antibody binding sites. The advantages of the CEDIA immunoassay system over conventional homogeneous EIA's include a linear dose response curve and lower limits of detection of analytes in human body fluids. The demonstrated sensitivity achievable with CEDIA technology suggests further applications on a wide range of analytes including vitamins, hormones, drugs and cancer markers. A new variant of CEDIA technology leading to a single liquid reagent immunoassay useful for on-site testing has also been developed.

Inducers of erythroleukemic differentiation. Relationship of structure to activity among planar-polar compounds.

Hexamethylenebisacetamide is a potent inducer of erythroid differentiation in murine erythroleukemia cells. A series of chemical compounds structurally related to hexamethylenebisacetamide were tested for inducing activity including polymethylene chains terminally substituted with various combinations of carboxylate, amino, amide, or sulfoxide groups. Effective "dimerization" of dimethyl sulfoxide through a linear polymethylene chain increases its inducing activity by a magnitude similar to that observed when N-methylacetamide is effectively dimerized in such a manner. It was found that all potent inducing agents possess both a hydrophilic and hydrophobic portion of the molecule, as well as a planar portion. All are Lewis bases, possessing a free electron pair available for hydrogen bonding. The polymethylene chain joining functional groups must be flexible and must be 5 to 6 carbon atoms in length to achieve maximal activity. Introduction of triple or double (cis or trans) bonds into the polymethylene chain does not alter activity.