Assays for trichomonal and other hydrolases
View Patent ↗The release by trichomonads of a hydrolase that hydrolyzes a narrowly defined class of substrates at a low pH without interference from hydrolases that are unrelated to trichomoniasis is the basis for a selective diagnostic assay for trichomoniasis that measures hydrolysis of any of these substrates by vaginal fluid at a low pH. Selective assays for trichomoniasis are also obtained by removing particulate matter from a sample of vaginal fluid to extract a fraction devoid of particles greater than a selected size, and where desired, combining the extracted fraction with any of certain specified hydrolase inhibitors, then testing the fraction for enzymatic hydrolase activity. These qualities of trichomoniasis are the basis for a series of diagnostic tests and test devices that produce results that are detectable by visual and other means with a high degree of accuracy.
1. A test device for assaying a sample of vaginal fluid for the presence of a hydrolytic enzyme associated with Trichomonas vaginalis , said test device comprising
a strip of solid porous material that allows aqueous liquids to pass therethrough while retaining particulate matter greater than 20 microns in diameter; and
a conjugate comprising a chromogen covalently bonded to a substrate for said hydrolytic enzyme and releasable from said substrate by hydrolysis, said chromogen being a species that undergoes a detectable change upon hydrolytic release from said substrate, and said conjugate deposited in a region of said strip that is spatially separated from a sample application site on said strip by a length of said strip that is free of said conjugate, such that a sample of vaginal fluid applied to said application site migrates through said length to remove said particulate matter before reaching said conjugate.
2. A test device in accordance with claim 1 in which said solid porous material retains particulate matter greater than 10 microns in diameter.
3. A test device in accordance with claim 1 in which said solid porous material retains particulate matter greater than 1 micron in diameter.
4. A test device in accordance with claim 1 in which said chromogen is a member selected from the group consisting of indoxyl, chlorophenol red, and tetrabromophenolphthalein ethyl ester.
5. A test device in accordance with claim 1 in which said substrate is a peptide of 2 to 3 amino acids, the C terminus of which is bonded directly to said chromogen and is a member selected from the group consisting of lysine and arginine.
6. A test device in accordance with claim 5 in which the N-terminus of said peptide is protected against hydrolysis by an N blocking group selected from the group consisting of carbobenzoxy, benzoyl, t-butoxycarbonyl, and a D-amino acid.