Method for the quantitative determination of alpha-amylase

• US 4,172,765 A
• Filed: 09/14/1977
• Issued: 10/30/1979
• Est. Priority Date: 09/14/1977
• Status: Expired due to Term

• Current Assignee: Technicon Instruments Corporation (Bayer AG)
• Original Assignee: Technicon Instruments Corporation (Bayer AG)
• Inventors: Keyes, Melvin H.
• Primary Examiner(s): Shapiro, Lionel M.
• Application Number:
  US05/833,318
• Time in Patent Office: 776 Days
• Field of Search: 195/103.5 S, 195/103.5 C, 195/103.5 R, 195/4, 195/7, 195/11
• US Class Current: 435/14
• CPC Class Codes: C12M 21/18 Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• CPC Class Codes: C12M 23/58 Reaction vessels connected in series or in parallel combinations of bioreactors with other apparatus, C12M43/00
• CPC Class Codes: C12M 41/32 of substances in solution
• CPC Class Codes: C12Q 1/40 involving amylase
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

This invention relates to a method and apparatus for rapid quantitative determination of α-amylase in aqueous samples such as blood serum, etc. The method comprises a flow-through of the sample through various immobilized reagents contained in sequential stages. A scavenger stage initially removes glucose originally present in the sample and comprises immobilized glucose oxidase and catalase. The glucose-free sample flows through an immobilized starch stage substrate preferably containing a high percentage of amylose to quantitatively react with the α-amylase in the sample and produce oligosaccharides. The sample with the oligosaccharides flows through a glucose-generating stage containing immobilized glucoamylase which converts the oligosaccharides to glucose. The glucose-containing sample enters a detection stage wherein the glucose is converted to H₂ O₂ by flowing through immobilized glucose oxidase, and the H₂ O₂ produced is quantitatively detected in detection means such as a polarographic cell. The immobilized reagents are preferably contained in an apparatus which comprises at least one column in combination with the detection means and a readout means.

Claims

1. A method for the quantitative determination of the pancreatic and salivary α

-amylases in an aqueous solution sample containing glucose as a contaminant comprising the steps of passing said sample through a scavenger stage containing immobilized scavenger reagents capable of reacting with and removing said glucose as a sample contaminant in the said determination, passing said glucose-free sample through a substrate stage comprising an immobilized starch reagent capable of reacting quantitatively with said α

-amylases to produce oligosaccharides, passing said oligosaccharides-containing sample through a glucose-generating stage comprising an immobilized glucose-generating reagent capable of reacting said oligosaccharides to a glucose reaction product, passing said glucose reaction product containing sample to a detection stage comprising an immobilized reagent capable of reacting with said glucose reaction product to generate hydrogen peroxide, determining the amount of said hydrogen peroxide generated in detection means, and relating the detection reading from said detection means to the quantitative amount of α

-amylases contained in said sample.

2. The method of claim 1, wherein said aqueous solution sample is diluted with an aqueous buffered diluent.

3. The method of claim 1, wherein said scavenger reagents comprise coimmobilized glucose oxidase and catalase.

4. The method of claim 1, wherein said scavenger stage comprises an immobilized catalase reagent subsequent to said scavenger reagents.

5. The method of claim 1, wherein said immobilized starch reagent is a starch high in amylose content.

6. The method of claim 1, wherein said starch reagent is potato starch.

7. The method of claim 1, wherein said glucose-generating reagent is glucoamylase.

8. The method of claim 1, wherein said glucose-generating reagent is maltase.

9. The method of claim 1, werein said oligosaccharides contain 2-6 monomers of glucose.

10. The method of claim 1, wherein said glucose reaction product in said detection stage is reacted with immobilized glucose oxidase.

11. The method of claim 1, wherein said detection stage comprises polarographic cell means.

12. The method of claim 1, wherein said detection stage comprises spectrographic means.

13. The method of claim 1, wherein said scavenger reagents comprise coimmobilized glucose oxidase and catalase on a hard inorganic porous support.

14. The method of claim 13, werein said porous support is glass.

15. The method of claim 1, wherein said substrate stage comprises an immobilized amylose starch on a porous refractory support.

16. The method of claim 15, wherein said porous support is alumina.

17. The method of claim 1, wherein said substrate stage comprises an amylose starch immobilized and cross-linked on porous alumina.

18. The method of claim 17, wherein said alumina is acid activated.

19. The method of claim 1, wherein said aqueous sample is buffered with an aqueous buffered diluent to pH in the range of about 5 to 7.

20. The method of claim 1, wherein said sample containing said glucose reaction product in said detection stage is passed in direct contact with detection means comprising polarographic electrodes and polarographically determining hydrogen peroxide in said reaction mixture while in direct contact with said electrodes.

21. The method of claim 1, wherein said sample is blood serum.

22. The method of claim 1, wherein said sample is urine.

39. The method of claim 1, wherein said glucose-generating stage comprises immobilized α

-amylase.

23. A method for the quantitative determination of the pancreatic and salivary α

-amylases contained in a sample of a body fluid wherein said fluid initially contains glucose as a contaminant in the quantitative determination comprising the steps of buffering said sample to a suitable pH value, passing said buffered sample through a scavenger stage containing coimmobilized reagents comprising glucose oxidase and catalase wherein said glucose is removed as a contaminant, passing said glucose-free sample through a substrate stage containing an immobilized starch reagent comprising a high amylose content on an alumina support wherein said starch reagent quantitatively reacts with the α

-amylases contained in said sample to produce oliqosaccharides, passing said oligosaccharides-containing sample through a glucose-generating stage containing an immobilized reagent comprising glucoamylase to product a glucose reaction product, passing said glucose-containing sample to a detection stage comprising an immobilized glucose oxidase reagent wherein said glucose reaction product is oxidized to generate hydrogen peroxide, determining with detection means the hydrogen peroxide generated and relating the amount of hydrogen peroxide generated to the quantitative amount of α

-amylases contained in said sample.

24. The method of claim 23, wherein said immobilized starch reagent is potato starch cross-linked on said alumina support.

25. The method of claim 23, wherein said sample is passed through said immobilized reagents of said several stages sequentially in a confined zone.

26. The method of claim 23, wherein said detection means comprises polarographic means.

27. The method of claim 23, wherein said detection means comprises spectrographic means.

40. The method of claim 23, wherein said glucose-generating stage comprises immobilized α

-amylase.

28. A method for the quantitative determination of the pancreatic and salivary α

-amylases contained in an aqueous solution sample containing glucose as a contaminant in the quantitative determination comprising the steps of buffering said sample to a suitable pH value, passing said buffered sample solution through a substrate stage containing an immobilized starch reagent comprising a high amylose content wherein said starch reagent quantitatively reacts with the α

-amylases contained in said sample to produce oligosaccharides, passing said oligosaccharides containing sample solution through a scavenger stage containing immobilized reagents wherein said glucose contaminant is removed, mixing said glucose-free, oligosaccharide-containing sample solution with a second buffer solution and passing said buffered sample solution through glucose-generating stage containing immobilized reagents, wherein said reagents produce a glucose reaction product from said oligosaccharides, passing said glucose-containing sample solution to a detection stage werein said glucose reaction product is reacted to generate hydrogen peroxide, passing said hydrogen peroxide containing sample solution through detection means and relating the amount of hydrogen peroxide generated to the quantitative amount of α

-amylases in said sample.

29. The method of claim 28, wherein said immobilized starch reagent is potato starch cross-linked on a porous support.

30. The method of claim 28, wherein said sample solution is passed through said scavenger reagents in several stages sequentially in a confined zone.

31. The method of claim 28, wherein said detection means comprises polarographic means.

32. The method of claim 28, wherein said detection means comprises sepctrographic means.

33. The method of claim 28, wherein said scavenger reagents comprise glucose oxidase and catalase.

34. The method of claim 28, wherein said glucose-generating reagent is glucoamylase.

35. The method of claim 28, wherein said glucose-generating reagent is maltase.

36. The method of claim 28, wherein said glucose reaction product is reacted with glucose oxidase.

37. The method of claim 28, wherein said substrate starch reagent is an amylose starch on alumina.

38. The method of claim 28, wherein said substrate starch reagent is an amylose starch immobilized and cross-linked on porous acid-activated alumina.

41. The method of claim 28, wherein said glucose-generating stage comprises immobilized α

-amylase.