Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1995-03-17
2001-06-26
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
Reexamination Certificate
active
06251621
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to methods of assaying for the presence of hydrolase activity (i.e., hydrolytic enzymes) in a sample or specimen. In particular, this invention relates to a method for detecting candidiasis by assaying for the presence of enzymatically active aspartic protease in a sample.
BACKGROUND OF THE INVENTION
Candida albicans
and other Candida species cause a number of common, medically important infections. Oral candidiasis, for example, is very common in patients with immunodeficiency. Moreover, vulvovagina candidiasis is one of the most frequent disorders in obstetrics and gynecology. It has been estimated that approximately three-quarters of all adult women suffer from at least one attack of this disease. (De Bernardis, et al.,
J. Clin. Microbiol
. 27(11):2598-2603 (1989)). As a result of its wide-spread occurrence, extensive amounts of research have gone into understanding the etiology of candidiasis.
Research has demonstrated that
Candida albicans
and other Candida species have an etiological involvement in human candidiasis, and it is now generally believed that candidiasis is caused primarily by the presence of
Candida albicans
. Further, there is now considerable evidence for a role of an aspartic protease or (interchangeably) acid proteinase as a virulence factor of
Candida albicans
. It is known that pure cultures of
Candida aibicans
secrete an aspartic protease when grown under precisely defined conditions. Similarly, it is known that pure strains of
Candida aibicans
isolated from women with symptomatic vulvovaginitis release this enzyme when they are subsequently grown in specifically defined culture medium.
Candida albicans
acid proteinase antigen, i.e., aspartic protease antigen, has been detected immunologic ally in the vaginal fluid of all women from which vulvovaginal
Candida albicans
was isolated. (De Bemnardis, et al., Abstract No. F-91 In: Abstracts of the Annual Meeting of the American Society for Microbiology, (Anaheim, Calif. 1990)). The concentration of
Candida albicans
acid proteinase antigen, however, was significantly higher in patients with symptomatic vulvovaginal candidiasis than in asymptomatic carriers. The vaginal fluid concentration of this antigen in women with candidiasis is approximately 176±15.2 ng/mL, whereas the vaginal fluid concentration of this antigen in women without isolation of
Candida albicans
, i.e., without clinical candidiasis, was less than 2 ng/ml. Asymptomatic
Candida albicans
carriers had intermediate antigen levels (94±18.5 ng/ml). These findings are a strong indication that acid proteinase (i.e., aspartic protease) is involved in the pathogenesis of vulvovaginal candidiasis.
Candida albicans
aspartic protease, however, is known to be unstable at body temperatures. Moreover, detection of the aspartic proteinase antigen immunologically did not indicate whether the enzyme was present in an enzymatically active form.
Candida albicans
acid proteinase is an extracellular aspartic protease. Aspartic proteases are one of the major classes of proteases. They contain one or more key aspartic acid residues which are required for activity.
Candida albicans
aspartic protease has a broad protein substrate specificity which includes, for example, albumin, hemoglobin, casein, immunoglobin A, and many other proteins. This enzyme performs optimally under acidic conditions (i.e., pH 2.5-5.5), and it is rapidly inactivated at a high pH (i.e, at pH 7.5).
Candida albicans
aspartic protease is strongly inhibited by pepstatin, but it is not inhibited by thiol reagents, chelators or serine protease inhibitors.
Many pharmaceutical companies and leading academicians are studying aspartic protease inhibitors for potential therapeutic use. Their efforts, however, are made difficult due to a lack of a suitable enzyme assay for aspartic proteases. While simple colorimetric assays are available for some serine, thiol, metallo, acid, and alkaline proteases and peptidases, they are not available for aspartic proteases. The substrate specificity of this particular class of enzymes requires the presence of several hydrophobic amino acids. This property has greatly hindered the search for simple synthetic chromogenic substrates because the hydrophobic amino acids which serve as the substrate for aspartic proteases are notoriously difficult to dissolve in water. As a result, chromogenic substrates for aspartic protease are not commercially available, are difficult to synthesize and characterize, and are poorly water soluble. The net effect of these limitations is that the enzyme activity as defmed by these chromogenic substrates is extremely low and thus, colorimetric assays for aspartic protease are quite insensitive. Similarly, fluorogenic substrates have been described for aspartic proteases, but they, too, are of limited utility. First, as previously mentioned, the substrate specificity of this particular class of enzymes requires the presence of several hydrophobic amino acids, rendering the substrates relatively insoluble. Second, at the low achievable concentrations of these substrates, the fluorogenic substrates are hydrolyzed very slowly and thus, fluorogenic assays are time-consuming. In addition, many biological specimens contain fluorescent materials which can interfere with fluorogenic assays for aspartic proteases.
Due to the lack of suitable colorimetric or fluorogenic assays for the detection of aspartic proteases, ultraviolet (UV) spectrophotometric assays are generally used to assay for the presence of this particular class of enzymes. In typical UV spectrophotometric assays, aspartic protease is added to a solution of protein (such as, for example, hemoglobin or albumin) and the mixture is incubated at 30-37° C. for 0.5 to 4 hours. After incubation, cold, concentrated trichloroacetic acid (TCA) is added to the chilled incubation mixture to precipitate the undigested protein, leaving ultraviolet light absorbing peptides in solution. Finally, the precipitated, undigested protein is pelleted by centrifugation for approximately 1 hour at refrigerated temperatures, the supernatant aspirated, and its Optical Density at 280 nm is determined to reflect the amount of protein hydrolysis. Although this assay can be used for the detection of aspartic proteases, it is both time-consuming and laborious.
To date, therefore, no convenient, simple, on-site assay has been developed for detecting the presence of enzymatically active aspartic proteases. Accordingly, the present invention is directed to a method of assaying for the presence of enzymatically active aspartic proteases which overcomes the problems and disadvantages of the prior art. Further, the methods of the present invention are also useful for assaying for the presence of other known hydrolytic enzymes, i.e., hydrolases.
SUMMARY OF THE INVENTION
It has now been discovered that enzymatically active
Candida albicans
aspartic protease is present in the vaginal fluid of women with vulvovaginal candidiasis. It has further been discovered that the presence of enzymatically active aspartic protease in a sample or specimen can serve as a marker for the detection and diagnosis of candidiasis. Accordingly, a method has now been developed for detecting candidiasis by assaying for the presence of enzymatically active aspartic protease in a sample.
In this method, a sample, e.g., vaginal fluid, is contacted with a solid support. The solid support with which the sample is contacted has a reporter enzyme (i.e., a signal generating enzyme) immobilized thereon. The reporter enzyme is immobilized on the solid support in a manner such that it is released from the solid support upon action of the enzymatically active aspartic protease if the enzymatically active aspartic protease is, in fact, present in the sample. The sample after having been contacted with the solid support is combined with an indicator. The indicator is any chemical species which is susceptible to a visible or detectable change (such as, for example, a c
Andreasen Terrence J.
Churhuri Aulena
Lawrence Paul J.
Gitomer Ralph
Litmus Concepts Inc.
Townsend and Townsend / and Crew LLP
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