Recombinant bacterial alkaline phosphatase as an immunodiagnostic enzyme.
The high turnover number of calf alkaline phosphatase (CAP) is one compelling reason for selecting it as the label in many enzyme immunoassays (EIA's). CAP's usefulness, however, is limited by its inherently low thermal stability which is even further compromised during the chemical preparation of enzyme: antibody conjugates. Bacterial alkaline phosphatase (BAP) could be an attractive alternative to CAP in view of the former's extreme thermotolerance at temperatures as high as 95 degrees C. BAP has not been commonly used in EIA's however, because of its low to moderate catalysis rate. Site-directed mutagenesis was used to overcome BAP's low enzymatic activity and create a protein possessing two desired characteristics: high thermostability and high specific activity. A3-35 fold increased activity over wild-type BAP was obtained in ten different recombinant (r)BAP's via introductions of single-point mutations. The turnover number of the most active mutant, D101S, was shown to be only 1.7-times lower than CAP. This dramatic improvement enables rBAP (D101S) to compete with CAP as a viable alternative label in EIA's. The thermostability of all ten rBAP remained significantly higher than CAP although none were as thermostable as the native BAP. Enzyme:antibody conjugates were prepared with the recombinant enzymes and compared to similarly prepared CAP:antibody conjugates with different Abbott IMx assay protocols and reagents. Excellent correlation between standard curves generated with CAP- and rBAP-containing conjugates were obtained. Furthermore, this correlation was obtained using concentrations of rBAP that were only two times greater than that of CAP. The thermostabilities of the conjugates were also evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)
Tomazic-Allen
S J
SJ
Abbott Diagnostic Division, Abbott Laboratories, IL.
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D004795:Enzyme Stability; D007124:Immunoenzyme Techniques; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D000469:Alkaline Phosphatase