Alpha-amylase is found primarily in the pancreas and salivary glands. When released in the digestive tract, the enzyme hydrolyses starch. Alpha-amylase determinations are useful in the diagnosis of diseases of the pancreas and parotids. Elevated serum levels are associated with acute pancreatitis and other pancreatic disorders, as well as mumps and bacterial parotitis. Decreased serum values may be found with liver diseases, such as hepatitis and obstructive jaundice, and liver tumors or abscesses.
Historically, methods for determining alpha-amylase in serum have included viscosimetric, turbidimetric, iodometric, and reductometric technology. With these methodologies, reaction times are long, endogenous glucose tends to interfere, reaction colors are unstable, and reproducibility is poor. Recently, assay systems for the determination of alpha-amylase have been developed.
Such assay systems for alpha-amylase typically include a reagent comprising a polysaccharide or oligosaccharide substrate with a label, e.g., a chromogen unit, attached. The substrate is hydrolyzed by alpha-amylase to form one or two smaller oligosaccharides. The reagent further comprise one or more enzymes which further hydrolyze the smaller oligosaccharides to free the label unit which can then be detected spectrophotometrically.
Such assay reagents enable rapid and accurate determinations of alpha-amylase compared to historical methodologies. However, the stability of such reagents is poor. Consequently, assay reagents are generally stored in a lyophilized state and must be reconstituted prior to use. Once reconstituted, the shelf life is generally one to fourteen days. Moreover, such reagents tend to give variable and often undesirably high background levels which adversely affect the consistency and accuracy of this system.
The above-identified patent applications disclose a stable, single-liquid alpha-amylase assay reagent for the rapid determination of the alpha-amylase in biological fluids. The assay reagent comprises an aqueous solution substantially free of alpha-amylase and/or alpha-amylase activity containing at least one substrate which is cleavable directly or indirectly by alpha-amylase to produce a detectable change in the reaction mixture. The detectable change may be the production or elimination of a detectable component. Such components may be detected by any suitable means including optical, electrochemical and thermochemical means.
In a preferred embodiment of the invention, the reagent comprises a polysaccharide or long-chain oligosaccharide substrate having a label attached at the reducing end. The substrate is hydrolyzable by alpha-amylase to form short-chain oligosaccharides, at least one of which comprises the label. The reagent further comprises at least one exo-enzyme, and preferably a pair of exo-enzymes, current maltase and alpha- or beta-glucosidase, which further hydrolyses the oligosaccharides to free the label which is then detectable spectrophotometrically. The rate at which the free label is formed provides a direct indication of the concentration of alpha-amylase in the biological fluid.
The alpha-amylase reagent is made substantially free of alpha-amylase by utilizing sterile water and purified reagents, and by passing the exo-enzymes and substrate, either individually or in combination, through a filter having a pore size of not more than about 0.2 micron to remove alpha-amylase-producing bacteria. Elimination of alpha-amylase from the reagent eliminates the consumption of the substrate during storage and hence stabilizes the reagent.
The alpha-amylase assay reagent is further stabilized by the inclusion of a polyol which retards the degradation of the exo-enzymes.
In commercial reagent we have employed as the exo-enzymes a mixture of a fairly high concentration of about 10 U/ml alpha-glucosidase (maltase) derived from yeast and 10 U/ml glucoamylase.