Claims for Patent: 5,910,422

Summary for Patent: 5,910,422

Title:	Stable single liquid alpha-amylase reagent
Abstract:	A stable, single liquid alpha-amylase assay reagent comprises a polysaccharide or long chain oligosaccharide substrate for alpha-amylase having an optically detectable label bonded to the reducing end glucose, by a bond cleavable by alpha- or beta-glucosidase, and a blocking group bonded to the terminal end glucose. The reagent further comprises beta-amylase and either alpha- or beta-glucosidase. Sorbitol is provided in an amount of about 5% to retard degradation of the enzymes. Zwitterionic buffers are also provided to stabilize the enzymes. In preparing the reagent, the enzymes and substrate are filtered through a 0.2 micron or less filter to remove alpha-amylase producing organisms.
Inventor(s):	Modrovich; Ivan E. (Camarillo, CA), Kwan; Shing S. (Camarillo, CA), Wortham; Leon C. (Canoga Park, CA)
Assignee:	Medical Analysis Systems, Inc. (Camarillo, CA)
Application Number:	07/700,996
Patent Claims:	1. A single liquid alpha-amylase reagent composition comprising an aqueous solution which is substantially free of alpha-amylase producing microorganisms which comprises: at least one substrate which is hydrolyzed when mixed with a sample of body fluid containing alpha-amylase to yield directly or indirectly by a reaction involving alpha-amylase to form a detectable label released to the reaction mixture, the rate of formation of said detectable label to the reaction mixture being proportional to the amount of alpha-amylase present in the sample and at least one exo-enzyme to cooperate with the alpha-amylase in the formation of the detectable label; and a compound selected from the group consisting of diols and polyols present in an amount sufficient to stabilize the exo-enzymes, said substrate being present in a concentration sufficient to prevent the substrate from limiting the rate of hydrolysis thereof , said reagent composition being stable against substrate and enzyme degradation for at least 6 months at 2 to 8.degree. C. 2. An alpha-amylase assay reagent according to claim 1 wherein the substrate comprises an optically detectable label cleavable directly or indirectly by alpha-amylase. 3. An alpha-amylase assay reagent according to claim 1 further comprising at least one enzyme and wherein the substrate reacts with alpha-amylase to produce a product which then reacts with the, enzyme to produce a detectable change in the reaction mixture. 4. A single liquid alpha-amylase reagent according to claim 1, wherein the compound is selected from the group consisting of diol and polyol is selected from the group consisting of ethylene glycol, polyethylene glycol, sorbitol, substituted glycols and mixtures thereof. 5. A single liquid alpha-amylase reagent comprising an aqueous solution substantially free of alpha-amylase which comprises: a substrate selected from the group consisting of polysaccharides and oligosaccharides, which substrate is hydrolyzable by alpha-amylase and having an optically detectable label, bonded to the substrate which is cleavable by alpha-glucosidase or beta-glucosidase, to the reducing end glucose of the substrate and a blocking group bonded to the terminable end glucose of the substrate, said alpha-amylase releasing the group from substrate label at a rate proportional to alpha-amylase concentration in a sample to enable formation of the label, said substrate present in a quantity sufficient not to limit the rate of hydrolysis; an exo-enzyme capable of cleaving the bond between the label and the reducing end glucose of the substrate selected from the group consisting of alpha-glucosidase and beta-glucosidase; and a compound selected from the group consisting of diols and polyols present in an amount sufficient to retard the degradation of the enzymes in the reagent said alpha-amylase reagent being stable for at least six months at 2 to 8.degree. C. 6. An alpha-amylase reagent according to claim 5 wherein the diol or polyol is selected from the group consisting of ethylene glycol, polyethylene glycol, sorbitol, and mixtures thereof. 7. An alpha-amylase assay reagent according to claim 5 wherein the diol or polyol is present in an amount of from about 10 to about 300 grams per liter of the reagent. 8. An alpha-amylase assay reagent according to claim 7 wherein the diol or polyol is present in an amount of about 30 to about 70 grams per liter of the reagent. 9. An alpha-amylase assay reagent according to claim 5 further comprising at least one buffer capable of maintaining the reagent at a pH of from about 4 to about 10. 10. An alpha-amylase assay reagent according to claim 9 wherein the buffer maintains the reagent at a pH of from about 6.5 to about 7.5. 11. An alpha-amylase assay reagent according to claim 9 wherein the buffer is a zwitterionic buffer capable of stablizing at least one of the enzymes of the reagent. 12. An alpha-amylase assay reagent according to claim 11 wherein the zwitterionic buffer is present in an concentration of from about 0.01 to about 1.0 moles per liter. 13. An alpha-amylase assay reagent according to claim 11 wherein the zwitterionic buffer is selected from the group consisting of MOPS, HEPES, TAPS, TAPSO and mixtures thereof. 14. An alpha-amylase assay reagent according to claim 13 wherein the zwitterionic buffer is MOPS. 15. An alpha-amylase assay reagent according to claim 5 wherein at least the enzymes have been passed through a filter having a pore size sufficiently small to retain substantially all alpha-amylase producing microorganisms. 16. An alpha-amylase assay reagent according to claim 15 wherein the filter has a pore size of no more than about 0.2 micron. 17. An alpha-amylase assay reagent according to claim 5 wherein the enzymes and the substrate have been passed through a filter having a pore size sufficiently small to retain substantially all alpha-amylase producing microorganisms. 18. An alpha-amylase assay reagent according to claim 17 wherein the filter has a pore size of no more than about 0.2 microns. 19. An alpha-amylase assay reagent according to claim 5 further comprising at least one antimicrobial agent. 20. An alpha-amylase assay reagent according to claim 19 wherein the antimicrobial agent is selected from the group consisting of CTMA bromide, bactrim, sodium azide, and mixtures thereof. 21. A method for preparing a stable, single liquid alpha-amylase assay reagent comprising: introducing at least one buffer into sterile water to provide a buffer solution having a pH of about 6.5 to about 7.5; introducing into the buffer solution a polysaccharide or oligosaccharide substrate for alpha-amylase having an optically detectable label bonded, by a bond cleavable by alpha-glucosidase or beta-glucosidase, and a blocking group bonded to each terminal end glucose of the substrate; beta-amylase; an exo-enzyme capable of cleaving the bond between the label and the substrate selected from the group consisting of alpha-glucosidase and beta-glucosidase; and at least one diol or polyol present in an amount sufficient to retard the degradation of the enzymes in the reagent; and passing the substrate and enzyme containing solution through a filter having a pore size sufficiently small to remove substantially all alpha-amylase producing microorganisms. 22. A method according to claim 21 wherein the diol or polyol is introduced in an amount of from about 10 to about 300 grams per liter of the buffer solution. 23. A method according to claim 22 wherein the diol or polyol is introduced in an amount of about 30 to about 70 grams per liter of the buffer solution. 24. A method according to claim 21 wherein the buffer is zwitterionic buffer capable of stabilizing at least one of the enzymes of the reagent. 25. A method according to claim 24 wherein the zwitterionic buffer is introduced in a concentration of from about 0.01 to about 1.0 moles per liter. 26. A method according to claim 24 wherein the zwitterionic buffer is selected from the group consisting of MOPS, HEPES, TAPS, TAPSO and mixtures thereof. 27. A method according to claim 25 wherein the zwitterionic buffer is MOPS. 28. A method according to claim 21 further comprising introducing into the buffer solution at least one antimicrobial agent. 29. A method according to claim 28 wherein the antimicrobial agent is selected from the group consisting of CTMA bromide, bactrim, sodium azide, and mixtures thereof. 30. A method for preparing a stable, single liquid alpha-amylase assay reagent comprising: preparing a buffer solution having a pH of from about 6.5 to about 7.5 comprising at least one buffering agent and sorbitol; introducing into a first portion of the buffer solution polysaccharide or oligosaccharide substrate for alpha-amylase having an optically detectable label bonded, by a bond cleavable by alpha-glucosidase or beta-glucosidase, to the reducing end glucose of the substrate and a blocking group bonded to the terminal end glucose of the substrate; introducing into a second portion of the buffer solution beta-amylase and an exo-enzyme capable of cleaving the bond between the label and the reducing and glucose of the substrate selected from the group consisting of alpha-glucosidase and beta-glucosidase; and filtering each of the first and second portions of the buffer solution through a filter having a pore size of no greater than about 0.2 microns; and mixing the first and second portions of the buffer solution. 31. A method according to claim 30 wherein sorbitol is present in an amount of from about 10 to about 300 grams per liter of the buffer solution. 32. A method according to claim 30 wherein sorbitol is present in an amount of from about 30 to about 70 grams per liter of the reagent. 33. A method according to claim 30 wherein the buffer is a zwitterionic buffering agent capable of stabilizing at least one of the enzymes of the reagent. 34. A method according to claim 33 wherein the zwitterionic buffering agent is present in a concentration of from about 0.01 to about 1.0 moles per liter. 35. A method according to claim 33 wherein the zwitterionic buffer is selected from the group consisting of MOPS, HEPES, TAPS, TAPSO and mixtures thereof. 36. A method according to claim 35 wherein the zwitterionic buffering agent is MOPS. 37. A method according to claim 36 wherein the antimicrobial agent is selected from the group consisting of CTMA bromide, bactrim, sodium azide, and mixtures thereof. 38. A method according to claim 30 wherein the buffer solution further comprises at least one antimicrobial agent. 39. A stable, single liquid alpha-amylase reagent comprising an aqueous solution substantially free of alpha-amylase producing microorganisms which comprises: a polysaccharide or oligosaccharide substrate hydrolyzable by alpha-amylase and having an optically detectable label bonded, by a bond cleavable by alpha-glucosidase or beta-glucosidase, to the reducing end glucose of the substrate and a blocking group bonded to the terminal end glucose of the substrate, said substrate being present in a concentration of at least 2 mg/ml of reagent and hydrolyzed in the presence of alpha-amylase to yield the label at a rate proportional to the concentration of alpha-amylase in a biological fluid; beta-amylase in a concentration of about 5 KIU/L; an exo-enzyme capable of cleaving the bond between the label and the reducing end glucose of the substrate selected from the group consisting of alpha-glucosidase and beta-glusidase, said exo-enzymes being present in a concentration of about 12.5 KIU/L; a polyol present in an amount of from about 10 to 300 grams per liter and sufficient calcium chloride in a concentration of about 5 mM and to retard said reagent buffered by a zwitterionic buffer to a pH of from about 4 to about 10 and being stable for at least 6 months at 2 to 8.degree. C. 40. An alpha-amylase assay reagent according to claim 39 wherein the polyol is sorbitol. 41. An alpha-amylase assay reagent according to claim 40 wherein the sorbitol is present in an amount of about 30 to 70 grams per liter of the reagent. 42. An alpha-amylase assay reagent according to claim 41 in which the buffer maintains the reagent at a pH of from about 6.5 to about 7.5. 43. An alpha-amylase assay reagent according to claim 42 the substrate is p-nitrophenylmaltoheptaoside. 44. An alpha-amylase assay reagent according to claim 42 wherein the enzymes and the substrate have been passed through a filter having a pore size no more than about 0.2 micron and sufficiently small to retain substantially all alpha-amylase producing microorganisms. 45. An alpha-amylase assay reagent according to claim 44 further comprising at least one antimicrobial agent. 46. An alpha-amylase assay reagent according to claim 45 wherein the antimicrobial agent is selected from the group consisting of CTMA bromide, bactrim, sodium azide, and mixtures thereof. 47. An alpha-amylase assay reagent according to claim 39 wherein the polyol is present in an amount of about 30 to about 70 grams per liter of the reagent. 48. An alpha-amylase assay reagent according to claim 39 wherein the zwitterionic buffer is present in an concentration of from about 0.01 to about 1.0 moles per liter. 49. An alpha-amylase assay reagent according to claim 45 wherein the zwitterionic buffer is selected from the group consisting of MOPS, HEPES, TAPS, TAPSO and mixtures thereof. 50. An alpha-amylase assay report reagent according to claim 39 wherein the zwitterionic buffer is selected from the group consisting of MOPS, HEPES, TAPS, TAPSO and mixtures thereof. 51. An alpha-amylase assay reagent according to claim 39 wherein the substrate is p-nitrophenylmaltoheptaoside. 52. An alpha-amylase assay reagent according to claim 39 wherein at least the enzymes have been passed through a filter having a pore size sufficiently small to retain substantially all alpha-amylase producing microorganisms. 53. An alpha-amylase assay reagent according to claim 52 wherein the filter has a pore size of no more than about 0.2 micron. 54. An alpha-amylase assay reagent according to claim 39 further comprising at least one antimicrobial agent. 55. An alpha-amylase assay reagent according to claim 54 wherein the antimicrobial agent is selected from the group consisting of CTMA bromide, bactrium, sodium azide, and mixtures thereof.

Details for Patent 5,910,422

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2016-06-08