Enzymes used in activity modulation assays

The previously discussed enzymes may all be used for solid-phase AM assays. For homogeneous AM-type EIA, lysozyme, malate dehy-rogenase (MDase), glucose-6-phosphate dehydrogenase (GPDase), and ribonuclease A (RNase A), are used in addition to BGase. 

Lysozyme (iV-acetylmuramide glycano-hydrolase, EC 3.2.1.17) from hen-egg white was the first enzyme used in homogeneous EIA (Ru-benstein et al., 1972) and is employed for a number of assays (Table 10.15). 

Lysozyme is used in rapid urine assays (total assay time 60 sec), but has some important limitations. Sometimes urine samples are positive for lysozyme and blanks should be run for all positive samples. These assays may also be affected by the pH, salt concentrations, and lysozyme inhibitors in urine. Blood and serum have high lysozyme levels. Detection limits are given at 95% confidence levels of semiquantitative tests (Ka-bakoff and Greenwood, 1982). 

One of the 6 lysyl residues (residue 97) of lysozyme is near the active site. Conjugation of a hapten to this group does not affect the activity of the enzyme, but incubation of the enzyme-hapten conjugate with the specific anti-hapten antibody prior to the addition of the bulky substrate, produces serious inhibition. The smaller Fab is not as effective for inhibition as complete IgG. 

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