Exclusively from Immunalysis, SEFRIA™ reagents are the next-generation β-galactosidase enzyme-based immunoassay in a liquid stable, ready to use format. This advanced technology is ultra-sensitive, enabling lower cutoffs, and at the same time, enhanced precision. SEFRIA reagents are designed for use on routine chemistry analyzers.
Principle of the procedure:
The SEFRIA™ technology is based on artificial fragments of the E. coli enzyme β-galactosidase. A mutant enzyme, termed Enzyme Acceptor (EA), is created by deletion of a short sequence in the amino-terminal region of the sequence. EA is inactive, but can combine with peptides, termed Enzyme Donors (ED’s), containing the deleted sequence, to form active β-galactosidase. This process is termed complementation, and the active enzyme formed as a result can be measured by hydrolysis rate of a chromogenic substrate such as chlorophenolred β-D-galactopyranoside (CPRG).
The ED peptides can be modified by attachment of a derivative of the target drug, which does not interfere with the formation of active β-galactosidase. However antibodies to the target drug bind to the ED-drug conjugate, and block complementation.
The assay is based on the competition of the target drug in a urine sample with the ED-drug conjugate for the fixed amount of antibody binding sites. In the absence of the free drug in the sample, the antibody binds the ED-drug conjugate, resulting in inhibition of enzyme formation. As the target drug concentration in the sample increases, ED-drug becomes available for complementation, creating a dose response relationship between the target drug concentration in the urine and enzyme formation. The β-galactosidase activity is determined spectrophotometrically at 570 nm by the conversion of CPRG (orange) to chlorophenol red (red) and galactose.