Assays for trichomonal and other hydrolases

Abstract

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  Trichomoniasis vaginalis , said test device comprising: 
 a flow passage having a defined application site for application of said sample;    a solid porous material, retained within said flow passage, that retains particulate matter greater than 20 microns in diameter while allowing aqueous liquids to pass;    an indicator deposited in solid form in said flow passage, said indicator being soluble in an aqueous liquid and producing a detectable change when contacted with an activated reagent; and    a conjugate deposited in solid form in said flow passage, said conjugate comprising said reagent covalently bonded to a substrate for said hydrolytic enzyme, said reagent releasable from said substrate by enzymatic hydrolytic cleavage action and thereby activated;    at least one of said indicator and said conjugate being spatially separated from said application site by a distance sufficiently great that a detectable change in said indicator is caused only by hydrolytic activity of said hydrolase in a fraction of said sample remaining after said particulate matter has been removed from said sample by said solid porous material.    
     
     
         2 . A test device in accordance with  claim 1  in which said application site and said solid porous material define a flow direction of said sample within said device, said indicator is deposited in a first location along said flow direction and said conjugate is deposited in a second location along said flow direction downstream of said first location.  
     
     
         3 . A test device in accordance with  claim 1  in which said solid porous material retains particulate matter greater than 10 microns in diameter.  
     
     
         4 . A test device in accordance with  claim 1  in which said solid porous material retains particulate matter greater than 1 micron in diameter.  
     
     
         5 . A test device in accordance with  claim 1  further comprising a hydrolase inhibitor deposited in solid form in said flow passage, said hydrolase inhibitor inhibiting hydrolases other than hydrolases characteristic of  Trichomoniasis vaginalis.    
     
     
         6 . A test device in accordance with  claim 2  further comprising a hydrolase inhibitor deposited in solid form in admixture with said indicator in said first location.  
     
     
         7 . A test device in accordance with  claim 5  in which said hydrolase inhibitor is a member selected from the group consisting of antipain, chymostatin, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, the polypeptide Lys-Pro-Gln-Leu-Trp-Pro, the polypeptide Arg-Lys-Asn-Val-Tyr, and the dipeptide Lys-Pro.  
     
     
         8 . A test device in accordance with  claim 1  in which said substrate is a peptide of 1 to 6 amino acids, the C-terminus of which is bonded directly to said reagent and the C-terminal amino acid is a member selected from the group consisting of lysine and arginine.  
     
     
         9 . A test device in accordance with  claim 1  in which said substrate is a peptide of 2 to 3 amino acids.  
     
     
         10 . A test device in accordance with  claim 1  in which the N-terminus of said peptide is protected against hydrolysis by an N-blocking group.  
     
     
         11 . A test device in accordance with  claim 10  in which said N-blocking group is a member selected from the group consisting of carbobenzoxy, benzoyl, t-butoxycarbonyl, and a D-amino acid.  
     
     
         12 . A test device in accordance with  claim 1  in which said reagent is a member selected from the group consisting of 4-methoxy-2-naphthylamine, beta.-naphthylamine, 7-amino-4-methylcoumarin, .alpha.-naphthol, .beta.-naphthol, 3-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthalenesulfonic acid, 1-naphthol-3,6-disulfonic acid, 6-bromo-2-naphthol, 6-hydroxy-2-naphthyl disulfide, and 4-hydroxy-1-naphthalenesulfonic acid, and said indicator is a diazonium dye.  
     
     
         13 . A test device in accordance with  claim 1  in which said conjugate is a member selected from the group consisting of carbobenzoxy-L-valine-L-argi-nine-4-methoxy-2-naphthylamine, carbobenzoxy-L-arginine-L-arginine-4-metho-xy-2-naphthylamine, carbobenzoxy-L-arginine-L-arginine-L-arginine-4-methox-y-2-naphthylamine, carbobenzoxy-L-leucine-L-arginine-4-methoxy-2-naphthyla-mine, carbobenzoxy-L-valine-L-arginine-4-methoxy-2-naphthylamine, and D-valine-L-leucine-L-arginine-4-methoxy-2-naphthylamine, and said indicator is a member selected from the group consisting of p-dimethylaminocinnamaldehyde, diazonium dyes and tetrazonium dyes.  
     
     
         14 . A test device in accordance with  claim 1  in which said conjugate is a member selected from the group consisting of carbobenzoxy-L-arginine-L-ar-ginine-L-arginine-4-methoxy-2-naphthylamine and D-valine-L-leucine-L-argin-ine-4-methoxy-2-naphthylamine, and said indicator is p-dimethylaminocinnamaldehyde.  
     
     
         15 . A test device in accordance with  claim 2  further comprising a developing reagent deposited in solid form in said flow passage in a third location spaced apart from said first and second locations, and means for manually placing said third location in contact with said second location.  
     
     
         16 . A test device in accordance with  claim 15  in which said indicator is p-dimethylaminocinnamaldehyde and said developing reagent is maleic acid.