Immunoassay for homocysteine

• US 6,063,581 A
• Filed: 09/14/1994
• Issued: 05/16/2000
• Est. Priority Date: 01/22/1992
• Status: Expired due to Term

• Current Assignee: Axis-Shield ASA
• Original Assignee: Axis-Shield ASA
• Inventors: Sundrehagen, Erling
• Primary Examiner(s): DUFFY, PATRICIA ANN
• Application Number:
  US08/256,367
• Time in Patent Office: 2,071 Days
• Field of Search: 435/7.1, 435/7.72, 435/7.9, 435/7.92, 435/7.93, 435/7.94, 435/15, 435/18, 435/975, 435/70.21, 436/518
• US Class Current: 435/7.1
• CPC Class Codes: C07H 15/14 attached to a sulfur, selenium or tellurium atom of a saccharide radical
• CPC Class Codes: C07H 15/26 Acyclic or carbocyclic radicals, substituted by hetero rings
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/5735 co-enzymes or co-factors, e.g. NAD, ATP
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6815 containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The invention relates to a method for assaying homocysteine in a sample such as blood, plasma or urine, which comprises the steps of contacting the sample with a homocysteine converting enzyme and at least one substrate for the enzyme other than homocysteine, and without chromatographic separation, assessing a non-labelled analyte selected from a homocysteine co-substrate and the homocysteine conversion products of the enzymic conversion of homocysteine by said enzyme.

Claims

1. An immunological method for indirectly assaying homocysteine in a sample, said method comprising the steps of:

(a) contacting said sample with S-adenosyl homocysteine hydrolase enzyme and adenosine or an analogue of adenosine, wherein said S-adenosyl homocysteine hydrolase converts said homocysteine into a non-labelled analyte, wherein said non-labelled analyte is S-adenosyl homocysteine or an analogue thereof; and

(b) determining the presence or amount of the non-labelled analyte without chromatographic separation by contacting said sample with an antibody which specifically binds with said non-labelled analyte and with a detectable hapten for said antibody other than said non-labelled analyte, and wherein determining the presence or amount of said non-labelled analyte is effected indirectly by determining the presence or amount of said detectable hapten either bound or not bound to said antibody, wherein the amount of the non-labelled analyte is directly proportional to the amount of homocysteine in said sample.

2. The method as claimed in claim 1 wherein said antibody is a monoclonal antibody.

3. The method as claimed in claim 1 wherein said antibody is a carrier matrix bound antibody.

4. The method as claimed in claim 1 wherein said sample is a blood, plasma or urine sample pre-treated with a disulphide bond cleaving reducing agent.

5. The method as claimed in claim 1 wherein the determination of the presence or amount of said non-labelled analyte is effected photometrically.

6. The method as claimed in claim 5 wherein said determination of the presence or amount of said non-labelled analyte is effected spectrometrically or calorimetrically.

7. The method as claimed in claim 5 wherein said determination of the presence or amount of said non-labelled analyte is effected turbidimetrically or nephelometrically.

8. The method as claimed in claim 5 wherein said determination of the presence or amount of said non-labelled analyte is effected using fluorescence polarization detection.

9. The method as claimed in claim 1 wherein as said detectable hapten is a compound of formula I ##STR16## wherein:

R₁ and R₂, which may be the same or different denote hydrogen atoms or OR₄ groups or R₁ and R₂ together denote an oxygen atom,

R₃ denotes an amino or carboxyl group, and

R₄ denotes a C_1-6 aliphatic group, or a salt or ester thereof.

10. The method as claimed in claim 1 wherein said detectable hapten is a labelled furanose 6-thioether, wherein the labelled moiety comprises a chromophore or fluorophore or a radioactive atom, and wherein the thioether moiety comprises a trimethylenethio moiety.

11. The method as claimed in claim 10 wherein as said labelled thioether is a labelled compound of formula I:

##STR17## wherein;

R₁ and R₂, which may be the same or different denote hydrogen atoms or OR₄ groups or R₁ and R₂ together denote an oxygen atom,

R₃ denotes an amino or carboxyl group, and

R₄ denotes a C_1-6 aliphatic group, or a salt or ester thereof.

12. The method as claimed in claim 1 wherein said enzyme was activated by contact with a reducing agent prior to contacting with said sample.

13. The method as claimed in claim 1 wherein said detectable hapten is selected from S-adenosyl homocysteine and a furanose 6-thioether labelled with a chromophore or a fluorophore.

14. The method of claim 1, wherein contacting comprises mixing the sample with adenosine and S-adenosyl homocysteine-hydrolase to form a first mixture, incubating the first mixture, contacting the incubated mixture with a fluorophore labelled compound selected from the group consisting of fluorophore labelled S-adenosyl-homocysteine and fluorophore labelled furanose 6-thioether to form a second mixture, the second mixture is then contacted with a monoclonal anti-S-adenoasyl-homocysteine antibody;

and wherein determining the presence of the non-labelled analyte comprises detecting the antibody-bound or non-antibody bound fluorophore labelled compound by fluorescence polarization.

15. The method of claim 1, wherein said contacting comprises mixing the sample with adenosine and S-adenosyl homocysteine-hydrolase to form a first mixture, incubating the first mixture, contacting the incubated mixture with a labelled compound selected from the group consisting of labelled S-adenosyl-homocysteine and fluorophore labelled furanose 6-thioether to form a second mixture, the second mixture is then contacted with a carrier matrix bound monoclonal anti-S-adenosyl-homocysteine antibody and washing the antibody bound carrier matrix;

and wherein determining the presence of the non-labelled analyte comprises photometrically detecting the antibody-bound labelled compound present on the washed carrier matrix.

16. The method of claim 15, wherein determining the presence of the non-labelled analyte further comprises contacting the antibody bound labelled compound on the washed carrier matrix with a substrate to generate a chromophore which is then photometrically detected.

17. An immunological method for indirectly assaying homocysteine in a sample, said method comprising the steps of:

(a) contacting said sample with S-adenosyl homocysteine hydrolase enzyme and S-adenosyl homocysteine wherein said S-adenosyl homocysteine hydrolase converts said S-adenosyl-homocysteine into a non-labelled analyte wherein said non-labelled analyte is adenosine; and

(b) determining the presence or amount of the non-labelled analyte without chromatographic separation by contacting said sample with an antibody which specifically binds with said non-labelled analyte and with a detectable hapten for said antibody other than said non-labelled analyte and wherein determining the presence or amount of said non-labelled analvte is effected indirectly by determining the presence or amount of said detectable hapten either bound or not bound to said antibody, wherein the amount of the non-labelled analyte is indirectly proportional to the amount of homocysteine in said sample.

18. The method as claimed in claim 17 wherein said antibody is a monoclonal antibody.

19. The method as claimed in claim 17 wherein said antibody is a carrier matrix bound antibody.

20. The method as claimed in claim 17 wherein said sample is a blood, plasma, or urine sample pre-treated with a disulphide bond cleaving reducing agent.

21. The method as claimed in claim 17 wherein the determination of the presence or amount of said analyte is effected photometrically.

22. The method as claimed in claim 21 wherein said determination of the presence or amount of said analyte is effected spectrometrically or colorimetrically.

23. The method as claimed in claim 21 wherein said determination of the presence or amount of said analyte is effected turbidimetrically or nephelometrically.

24. The method as claimed in claim 21 wherein said determination of the presence or amount of said analyte is effected using fluorescence polarization detection.

25. The method as claimed in claim 17 wherein said enzyme is SAH-hydrolase which has been activated by contact with a reducing agent prior to contacting with said sample.

26. The method as claimed in claim 17, wherein said detectable hapten is adenosine or an analogue thereof labelled with a chromophore or a fluorophore.

27. The method as claimed in claim 17 wherein said contacting comprising mixing the sample with S-adenosyl homocysteine and an S-adenosyl homocysteine-hydrolase to form a first mixture;

incubating the first mixture, contacting the incubated mixture with a fluorophore labelled compound selected from the group consisting of adenosine or an analogue of adenosine to form a second mixture;

the second mixture is then contacted with a monoclonal anti-adenosine or an antibody against an analogue of adenosine; and

the antibody-bound or non-antibody bound fluorophore labelled compound is detected by fluorescence polarization.

28. The method as claimed in claim 17 wherein said contacting comprises mixing the sample with S-adenosyl homocysteine and an S-adenosyl-homocysteine hydrolase to form a first mixture;

incubating the first mixture, contacting the incubated mixture with a labelled adenosine or a labelled analogue of adenosine to form a second mixture;

the second mixture is then contacted with carrier matrix bound monoclonal anti-adenosine antibody or an antibody against an analogue of adenosine;

the antibody carrier matrix is washed; and

the antibody-bound labelled compound is photometrically detected.

29. The method as claimed in claim 28 wherein the label of the antibody bound labelled compound is contacted with a substrate to generate a chromophore which is then photometrically assessed.