Method for measuring adenyl group-containing substances using heteropoly-acid

• US 5,610,016 A
• Filed: 03/30/1995
• Issued: 03/11/1997
• Est. Priority Date: 03/30/1994
• Status: Expired due to Fees

• Current Assignee: Mochida Pharmaceutical Company Limited
• Original Assignee: Mochida Pharmaceutical Company Limited
• Inventors: Shirakawa, Kamon, Sato, Naofumi
• Primary Examiner(s): Sisson, Bradley L.
• Assistant Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/413,279
• Time in Patent Office: 712 Days
• Field of Search: 436/94, 435/6, 435/968, 250/361, 536/243
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

The invention provides an assay by which detection and/or quantitation of adenyl group-containing substances can be performed simply, with high sensitivity and high signal-to-noise ratio. In particular, the method provides an assay in which the chemiluminescence of a chemiluminescent substance, made by reacting a glyoxal derivative with an adenyl group in the substance to be detected/quantitated in the presence of a heteropolyacid or a heteropolyacid salt, is measured.

Claims

1. A method for detecting the presence of or quantitating the amount of an adenyl group-containing substance which comprises:

i) deriving a chemiluminescent substance by reacting a compound represented by formula (1);

space="preserve" listing-type="equation">R.sup.1 --CO--R.sup.2 (

     1)

wherein R¹ is a member selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 1 to 12 carbon atoms, an alkynyl group having 1 to 12 carbon atoms, an aryl group having 1 to 18 carbon atoms and an aromatic heterocyclic group having 1 to 18 carbon atoms, wherein R¹ may be substituted or may form a condensed ring, and

wherein R² is an aldehyde group or a group represented by the formula (2);

space="preserve" listing-type="equation">--CH(XR.sup.3) (X'"'"'R.sup.4) (2)

wherein X and X'"'"' are independently an oxygen atom or a sulfur atom, and R³ and R⁴ are independently selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, an alkenyl group having 1 to 12 carbon atoms, an alkynyl group having 1 to 12 carbon atoms and an aryl group having 1 to 16 carbon atoms, wherein R³ and R² may be substituted or may form a condensed ring;

wherein R³ and R⁴ can optionally form a ring by binding;

with said adenyl group in the substance to be measured in the presence of a heteropolyacid or a salt thereof which is at least one compound selected from the group consisting of tungstosilicic acid, tungstophosphoric acid, tungstoarsenic acid, tungstogermanic acid, molybdosilicic acid, molybdophosphoric acid, molybdoarsenic acid, molybdogermanic acid, vanadophosphoric acid, and sodium, potassium and ammonium salts thereof, and

ii) measuring chemiluminescence of the chemiluminescent substance in a luminescence solvent, thereby detecting the presence of or quantitating the amount of said adenyl group-containing substance.

2. A method according to claim 1, wherein said heteropolyacid or salt thereof is at least one compound selected from the group consisting of tungstosilicic acid, tungstophosphoric acid, tungstoarsenic acid, molybdosilicic acid, molybdophosphoric acid, molybdoarsenic acid, and sodium molybdophosphoric acid.

3. A method according to claim 2, wherein said heteropolyacid or salt thereof is at least one compound selected from the group consisting of tungstosilicic acid, tungstophosphoric acid, molybdophosphoric acid, and sodium molybdophosphoric acid.

4. A method according to any one of claims 1 to 3, wherein said substance to be measured is adenine, adenosine, an adenosine phosphate compound, DNA or RNA.

5. A method according to claim 1,

wherein R¹ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, a phenyl group and an aromatic heterocyclic group having 1 to 8 carbon atoms, wherein R¹ may be substituted or may form a condensed ring, and

wherein R³ and R⁴ are independently groups which are selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, and a phenyl group, wherein R³ and R⁴ may be substituted or may form a condensed ring,

wherein R³ and R⁴ can optionally form a ring by binding.

6. A method according to claim 5, wherein said substance to be measured is adenine, adenosine, an adenosine phosphate compound, DNA or RNA.

7. A method according to claim 5, wherein said compound represented by formula (1) is a glyoxal derivative selected from the group consisting of methylglyoxal, methylglyoxal dimethylacetal, ethytglyoxal dimethylacetal, n-butylglyoxal dimethylacetal, n-octylglyoxal dimethylacetal, phenylglyoxal, phenylglyoxal dimethytacetal, p-methylphenylglyoxal and, p-fluorophenylglyoxal.

8. A method according to claim 7, wherein said substance to be measured is adenine, adenosine, an adenosine phosphate compound, DNA or RNA.

9. A method according to any one of claims 1 to 3, wherein chemiluminescence is measured by contacting said chemiluminescent substance with a reaction initiator in said luminescence solvent.

10. A method according to claim 9, wherein said luminescence solvent is at least one solvent selected from the group consisting of dimethylformamide, isopropanol, acetonitrile, dioxane, and dimethyl sulfoxide.

11. A method according to claim 1, wherein said compound of formula (1) is selected from the group consisting of n-alkyl glyoxal, phenylglyoxal, p-halophenylglyoxal, p-alkylphenylglyoxal, n-alkylglyoxal dimethylacetal, phenylglyoxal dimethylacetal, p-halophenylglyoXal dimethylacetal, and p-alkylphenylglyoxal dimethylacetal.

12. A method according to claim 3, wherein said compound of formula (1) is selected from the group consisting of n-alkyl glyoxal, phenylglyoxal, p-halophenylglyoxal, p-alkylphenylglyoxal, n-alkylglyoxal dimethylacetat, phenylglyoxal dimethylacetal, p-halophenylglyoxal dimethylacetal, and p-alkylphenylglyoxal dimethylacetal.

13. A method according to claim 3, wherein said compound of formula (1) is selected from the group consisting of methyl glyoxal, phenylglyoxal, methylglyoxal dimethylacetal, phenylglyoxal dimethylacetal, ethylglyoxal dimethylacetal, n-butylglyoxal dimethylacetal, p-methylphenylglyoxal, p-fluorophenylglyoxal and n-octylglyoxal dimethylacetal.