Claims
- 1. A composition for exhibiting sufficient color transition upon contacting a test sample to demonstrate the presence or concentration of D-.beta.-hydroxybutyrate in the test sample, said composition consisting essentially of:
- a) about 50 to about 5000 units of D-.beta.-hydroxybutyrate dehydrogenase;
- b) about 10 to about 500 mM of nicotinamide adenine dinucleotide;
- c) a disulfide reductase system comprising about 10 to about 200 mM of L-cystine and about 100 to about 2000 units of cystine reductase; and
- d) a sufficient amount of a thiol-responsive indicator dye to provide a measurable color transition in proportion to the amount of D-.beta.-hydroxybutyrate in the test sample.
- 2. The composition of claim 1 wherein the thiol-responsive indicator dye is an amide derivative of Ellman's reagent or an ester derivative of Ellman's reagent represented by the structural formula ##STR29## wherein the R moiety is the residue of an alcohol or 3-N-(3-dimethylaminopropyl)carboxamido-4-nitrophenyl disulfide.
- 3. The composition of claim 1 wherein the thiol-responsive indicator dye is a 2,2'-dipyridyl sulfide or a 4,4'-dipyridyl disulfide.
- 4. The composition of claim 3 wherein the 2,2'-dipyridyl disulfide is 2,2'-dithiobis(5-nitropyridine).
- 5. The composition of claim 1 wherein the thiol-responsive indicator dye is an isobenzothiazolone compound having the formula: ##STR30## wherein at least one of the R.sub.1 and R.sub.2 substituents is selected from the group consisting of nitro, arylazo, substituted arylazo, benzylideneamino and substituted benzylideneamino; and the R.sub.3 substituent is selected from the group consisting of alkyl, aryl, carboxyalkyl, hydroxyalkyl, aminoaryl, heteroaryl, carboxyheteroaryl, hydroxyheteroaryl, aminoheteroaryl, hydroxy, alkoxy, amino and substituted derivatives thereof.
- 6. The composition of claim 5 wherein when the R.sub.1 substituent is nitro, arylazo, substituted arylazo, benzylideneamino or substituted benzylideneamino, the R.sub.2 substituent is hydrogen.
- 7. The composition of claim 5 wherein when the R.sub.2 substituent is nitro, arylazo, substituted arylazo, benzylideneamino or substituted benzylideneamino, the R.sub.1 substituent is hydrogen.
- 8. The composition of claim 5 wherein the isobenzothiazolone compound includes R.sub.1 as nitro and R.sub.2 as hydrogen.
- 9. The composition of claim 5 wherein the isobenzothiazolone compound includes R.sub.2 as hydrogen and R.sub.1 is selected from the group consisting of phenylazo; 4-hydroxyphenylazo; 4-nitro-2-methylphenylazo; 2-hydroxy-1-naphthylazo; 2-hydroxy-5-methylphenylazo; 2-hydroxy-4-methyl-5-nitrophenylazo; 4-hydroxy-1-naphthylazo; 4-hydroxy-3-methyl-1-naphthylazo; 4-hydroxy-5-aza-1-naphthylazo; 2-amino-1-naphthylazo; 1-hydroxy-1-naphthylazo; 1-hydroxy-2-(N-dimethylaminopropylcarbamoyl)-4-naphthylazo; 3-N,N-dimethylaminopropylcarboxyamido-1-hydroxy-4-naphthylazo; 1-hydroxy-4-methoxy-2-naphthylazo; 2-hydroxy-3-carboxy-1-naphthylazo; 1-hydroxy-3,6-disulfonate-2-naphthylazo; 2,3-dihydroxy-1-naphthylazo; and 2-hydroxy-3,5-dimethyl-1-phenylazo.
- 10. The composition of claim 5 wherein the isobenzothiazolone compound includes R.sub.1 as 2,4-dinitrobenzylideneamino and R.sub.2 as hydrogen.
- 11. The composition of claim 5 wherein the isobenzothiazolone compound includes R.sub.1 as hydrogen and R.sub.2 as 2-hydroxy-1-naphthylazo or 4-hydroxy-1-phenylazo.
- 12. The composition of claim 5 wherein the isobenzothiazolone compound includes an R.sub.3 substituent which is selected from the group consisting of alkyl, aryl, heteroaryl, hydroxy, alkoxy and amino moieties.
- 13. The composition of claim 12 wherein the R.sub.3 substituent is substituted to include a moiety selected from the group consisting of aminoalkyl, aminoaryl, carboxyalkyl, carboxyaryl, hydroxyalkyl, hydroxyaryl and haloalkyl.
- 14. The composition of claim 5 wherein the R.sub.3 substituent is 3-dimethylaminopropyl.
- 15. The composition of claim 1 wherein the thiol-responsive indicator dye is selected from the group consisting of Ellman's reagent, 3-N-(3-dimethylaminopropyl)carboxamido-4-nitrophenyl disulfide, 2,2'-dithiobis(5-nitropyridine), N-3-(dimethylaminopropyl)-5-nitroisobenzothiazol-3-one, N-(3-dimethylaminopropyl)-5,7-dinitroisobenzothiazol-3-one, N-(3-dimethylaminopropyl)-5-(4-hydroxyphenylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(4-nitro-2-methylphenylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(2,4-dinitrobenzylideneamino)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-phenylazoisobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(2-hydroxy-1-naphthylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(2-hydroxy-5-methylphenylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(2-hydroxy-4-nitro-5-methylphenylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(4-hydroxy-1-naphthylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(4-hydroxy-3-methyl-1-naphthylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(4-hydroxy-5-aza-1-naphthylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(2-amino-1-naphthylazo)isobenzothiazol-3-one; N-(3-dimethylaminopropyl)-5-(4-methoxy-1-hydroxy-2-naphthylazo)isobenzothiazol-3-one; and combinations thereof.
Parent Case Info
This is a division of application Ser. No. 545,634, filed on Jun. 29, 1990 now U.S. Pat. No. 5,190,863.
US Referenced Citations (11)
Foreign Referenced Citations (1)
Number |
Date |
Country |
0140589 |
Aug 1985 |
EPX |
Non-Patent Literature Citations (2)
Entry |
"Direct Automated Assay Method for Serum or Urine Levels of Ketone Bodies,", Harano et al, 1985, pp. 177-188. |
"Development of Paper-Strip Test for 3-Hydroxybutyrate & its Clinical Application" Harano et al, 1984, pp. 481-485. |
Divisions (1)
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Number |
Date |
Country |
Parent |
545634 |
Jun 1990 |
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