Method and reagent for detecting a ligand in a sample

• US 5,821,061 A
• Filed: 09/06/1996
• Issued: 10/13/1998
• Est. Priority Date: 01/12/1995
• Status: Expired due to Fees

• Current Assignee: Toyoda Gosei Company Limited (Toyota Motor Corporation)
• Original Assignee: Toyoda Gosei Company Limited (Toyota Motor Corporation)
• Inventors: Yamamoto, Kazumi, Kawamura, Yoshihisa, Hattori, Shizuo, Teshima, Shinichi
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/709,434
• Time in Patent Office: 767 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/196, 435/21, 536/22.1, 536/25.32, 530/350
• US Class Current: 435/6.19
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12R 2001/07 Bacillus
• CPC Class Codes: G01N 2333/916 acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

A novel alkaline phosphatase and a method for the production of the alkaline phosphatase are described. A method for detecting a ligand in a sample and a method for analyzing the sequence of nucleic acid in a sample using the alkaline phosphatase are also described.

Claims

1. A method of detecting a ligand in a sample, comprising:

reacting a ligand in a sample with a substance bonded with a Bacillus alkaline phosphatase, the substance having a specific affinity for said ligand, so as to produce reaction products and unreacted substances;

separating the reaction products from the unreacted substances; and

measuring the activity of the alkaline phosphatase bonded with the reaction products or with the unreacted substances;

wherein the alkaline phosphatase has the following physical and chemical properties;

(1) the alkaline phosphatase catalyzes the following reaction;

space="preserve" listing-type="equation">orthophosphoric acid monoester+H.sub.2 O→

alcohol+orthophosphoric acid;

(2) the alkaline phosphatase is used in a buffer containing a Mg⁺⁺ salt and a Co⁺⁺ salt;

(3) the alkaline phosphatase is stable at least for 30 minutes when treated at pH 7.5 and 60°

C.;

(4) the alkaline phosphatase has a specific activity of at least 2,300 U/mg;

(5) the alkaline phosphatase has no sugar chain; and

(6) the alkaline phosphatase has a gel filtration molecular weight from 140,000 to 150,000 and a SDS-PAGE molecular weight from 65,000 to 67,000.

2. A method of detecting a ligand in a sample according to claim 1 in which the method of detecting a ligand in a sample is a method wherein an affinity reaction of the ligand in the sample with a substance having a specific affinity for said ligand is utilized and the activity of the alkaline phosphatase which is bonded with the substance by said reaction is measured or the activity of the alkaline phosphatase which is not bonded is measured.

3. A method of detecting a ligand in a sample according to claim 1 in which the alkaline phosphatase is bonded with a substance which has a specific affinity for the ligand.

4. A method of detecting a ligand in a sample according to claim 1 wherein the substance having a specific affinity for the ligand is bonded to a second substance and the second substance is bonded to a third substance, wherein the third substance is bonded to the alkaline phosphatase.

5. A method of detecting a ligand in a sample according to claim 1 in which the ligand is antigen, antibody, hormone, hormone receptor or nucleic acid.

6. A method of detecting a ligand in a sample according to claim 1 in which the affinity reaction of a ligand with a substance having a specific affinity for the ligand is an antigen-antibody reaction.

7. A method of detecting a ligand in a sample according to claim 1 in which the affinity reaction of a ligand with a substance having a specific affinity for the ligand is a hormone-hormone receptor reaction.

8. A method of detecting a ligand in a sample according to claim 1 in which the affinity reaction of a ligand with a substance having a specific affinity for the ligand is a nucleic acid hybridization reaction.

9. A method of detecting a ligand in a sample according to claim 1 in which any of the ligand and the substance having a specific affinity for said ligand is bonded with a solid phase.

18. The method according to claim 4 wherein the second substance is a biotin and the third substance is an avidin or wherein the second substance is an avidin and the third substance is an biotin.

19. The method according to claim 18 wherein a reaction binding the avidin with the biotin occurs simultaneous with or after the step of reacting the ligand in the sample with the substance having an affinity for the ligand.

10. A reagent for detecting a ligand in a biological sample containing a reagent for measuring an alkaline phosphatase and a ligand labeled with an alkaline phosphatase having the following physical and chemical properties or a specific bonding substance to a ligand labeled with an alkaline phosphatase having the following physical and chemical properties:

(1) the alkaline phosphatase catalyzes the following reaction;

space="preserve" listing-type="equation">orthophosphoric acid monoester+H.sub.2 O→

alcohol+orthophosphoric acid;

(2) the alkaline phosphatase is used in a buffer containing a Mg⁺⁺ salt and a Co⁺⁺ salt;

(3) the alkaline phosphatase is stable at least for 30 minutes when treated at pH 7.5 and 60°

C.;

(4) the alkaline phosphatase has a specific activity of at least 2,300 U/mg;

(5) the alkaline phosphatase has no sugar chain; and

(6) the alkaline phosphatase has a gel filtration molecular weight from 140,000 to 150,000 and a SDS-PAGE molecular weight from 65,000 to 67,000.

12. A reagent for detecting a ligand in a biological sample according to claim 10 or 11 in which the reagent for measuring the alkaline phosphatase is a 1,2-dioxetane compound or derivatives thereof, phenalenone-6-phosphate, benzophenalene-6-phosphate, 4-methylumbelliferone phosphate, p-nitrophenyl phosphate, 1-naphtholphthaleine phosphate, 5-bromo-4-chloro-3-indolyl phosphate or derivatives of those compounds.

13. A reagent for detecting a ligand in a biological sample according to claim 10 or 11 in which the ligand is antigen, antibody, hormone, hormone receptor or nucleic acid.

14. A reagent for detecting a ligand in a biological sample according to claim 10 or 11 in which the affinity reaction of a ligand with a substance having a specific affinity for the ligand is an antigen-antibody reaction.

15. A reagent for detecting a ligand in a biological sample according to claim 10 or 11 in which the affinity reaction of a ligand with a substance having a specific affinity for the ligand is a hormone-hormone receptor reaction.

16. A reagent for detecting a ligand in a biological sample according to claim 10 or 11 in which the affinity reaction of a ligand with a substance having a specific affinity for the ligand is a nucleic acid hybridization reaction.

17. A reagent for detecting a ligand in a biological sample according to claim 10 or 11 in which any of the ligand and the substance having a specific affinity for the ligand is bonded with a solid phase.

11. A reagent for detecting a ligand in a biological sample containing (I) a substance which has a specific affinity for a ligand to which avidin or biotin is bonded, (ii) an alkaline phosphatase having the following physical and chemical properties to which biotin or avidin is bonded and (iii) a substance for measuring an alkaline phosphatase:

(1) the alkaline phosphatase catalyzes the following reaction;

space="preserve" listing-type="equation">orthophosphoric acid monoester+H.sub.2 O→

alcohol+orthophosphoric acids;

(2) the alkaline phosphatase is used in a buffer containing a Mg⁺⁺ salt and a Co⁺⁺ salt;

(3) the alkaline phosphatase is stable at least for 30 minutes when treated at pH 7.5 and 60°

C.;

(4) the alkaline phosphatase has a specific activity of at least 2,300 U/mg;

(5) the alkaline phosphatase has no sugar chain; and

(6) the alkaline phosphatase has a gel filtration molecular weight from 140,000 to 150,000 and a SDS-PAGE molecular weight from 65,000 to 67,000.