Assays for amphetamine and methamphetamine using stereospecific reagents
View Patent ↗Methods, compositions and kits are disclosed. Enzyme conjugates of Formula I may be employed in assays for the determination of an amphetamine and/or a methamphetamine. Immunogenic conjugates of Formula I may be employed to prepare antibodies for an amphetamine and/or for a methamphetamine for use in assays for the determination of an amphetamine and/or a methamphetamine. The enzyme conjugates may also be employed to screen antibodies for use in such methods.
1. A compound of the formula:
wherein:
R 1 is H, lower alkyl, a protecting group,
R 2 is H, lower alkyl, a protecting group,
L is —(CH 2 ) t —X—(CH 2 ) v —Y— wherein X is C(O) or SO 2 , Y is a bond, S or —NR 3 wherein R 3 is H or lower alkyl, W is O, S, or NH, and t is an integer from 1 to 6 and v is an integer from 2 to 6,
Z is H, a protecting group, a poly(amino acid), a non-poly(amino acid) label moiety, a non-poly(amino acid) immunogenic carrier, or a functional group excluding thiol,
n is 1 when Z is other than a poly(amino acid) or, when Z is a poly(amino acid), n is an integer between 1 and the molecular weight of the poly(amino acid) divided by 500;
and salts thereof.
2. A compound according to claim 1 wherein R 1 is H and R 2 is H.
3. A compound according to claim 1 wherein R 1 is H and R 2 is lower alkyl.
4. A compound according to claim 3 wherein lower alkyl is methyl.
5. A compound according to claim 1 wherein Z is a poly(amino acid).
6. A compound according to claim 5 wherein said poly(amino acid) is an enzyme or a protein immunogenic carrier.
7. A compound of the formula:
wherein:
R 1 ′ is H, lower alkyl, a protecting group,
R 2 ′ is H, alkyl, a protecting group,
W′ is O, S or NR 3 wherein R 3 is H or lower alkyl,
X′ is C(O) or SO 2 ,
Y′ is bond, S or —NR 3 wherein R 3 is H or lower alkyl,
Z′ is H, a protecting group, a poly(amino acid), a non-poly(amino acid) label moiety, a non-poly(amino acid) immunogenic carrier, or a functional group,
t′ is an integer from 1 to 6 and v′ is an integer from 2 to 6,
n′ is 1 when Z′ is other than a poly(amino acid) or, when Z′ is a poly(amino acid), n′ is an integer between 1 and the molecular weight of the poly(amino acid) divided by 500;
and salts thereof.
8. A compound according to claim 7 wherein R 1 ′ is H and R 2 ′ is H.
9. A compound according to claim 7 wherein R 1 ′ is H and R 2 ′ is methyl.
10. A compound according to claim 7 wherein Z′ is a poly(amino acid).
11. A compound according to claim 7 , wherein said stereoisomeric mixture comprises at least 90% of one stereoisomeric form over the other.
12. A compound according to claim 11 wherein said stereoisomer has the formula:
13. A compound of the formula:
wherein:
R 1 ″ is H, lower alkyl, a protecting group,
R 2 ″ is H, lower alkyl, a protecting group,
W″ is O, S, or NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Y″ is bond, S or —NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Z″ is H, a protecting group, a poly(amino acid), a non-poly(amino acid) label moiety, a non-poly(amino acid) immunogenic carrier, or a functional group,
t″ is an integer from 1 to 6 and v″ is an integer from 2 to 6,
n″ is 1 when Z″ is other than a poly(amino acid) or, when Z″ is a poly(amino acid), n″ is an integer between 1 and the molecular weight of the poly(amino acid) divided by 500;
and salts thereof.
14. A compound according to claim 13 wherein R 1 ″ is H and R 2 ″ is H.
15. A compound according to claim 13 wherein R 1 ″ is H and R 2 ″ is methyl.
16. A compound according to claim 13 wherein Z″ is an enzyme.
17. A compound according to claim 16 wherein said enzyme is glucose-6-phosphate dehydrogenase.
18. A compound according to claim 13 wherein Z″ is a protein immunogenic carrier or a non-poly(amino acid) immunogenic carrier.
19. An antibody raised against a compound according to claim 18 .
20. A reagent system comprising a compound according to claim 16 , an antibody for amphetamine and/or an antibody for methamphetamine.
21. A reagent system comprising an antibody according to claim 19 and an enzyme conjugate of an amphetamine and/or an enzyme conjugate of methamphetamine.
22. A compound of the formula:
wherein:
R 1 ′″ is H, lower alkyl, a protecting group,
R 2 ′″ is H, lower alkyl, a protecting group,
W″ is O, S, or NR 3 ′″ wherein R 3 ′″ is H or lower alkyl,
Y′″ is a bond, S or —NR 3 ′″ wherein R 3 ′″ is H or lower alkyl,
Z′″ is H, a protecting group, a poly(amino acid), a non-poly(amino acid) label moiety, a non-poly(amino acid) immunogenic carrier, or a functional group,
t′″ is an integer from 1 to 6 and v′″ is an integer from 2 to 6,
n′″ is 1 when Z′″ is other than a poly(amino acid) or, when Z′″ is a poly(amino acid), n′″ is an integer between 1 and the molecular weight of the poly(amino acid) divided by 500;
and salts thereof.
23. A compound according to claim 22 wherein R 1 ′″ is H and R 2 ′″ is H.
24. A compound according to claim 22 wherein R 1 ′″ is H and R 2 ′″ is methyl.
25. A compound according to claim 22 wherein Z′″ is an enzyme.
26. A compound according to claim 25 wherein said enzyme is glucose-6-phosphate dehydrogenase.
27. A compound according to claim 22 wherein Z′″ is an antigen or a non-poly(amino acid) immunogenic carrier.
28. An antibody raised against a compound according to claim 27 .
29. A reagent system comprising a compound according to claim 25 , an antibody for amphetamine and/or an antibody for methamphetamine.
30. A reagent system comprising an antibody according to claim 28 and an enzyme conjugate of an amphetamine and/or an enzyme conjugate of methamphetamine.
31. A method for determining amphetamine and/or methamphetamine in a sample suspected of containing amphetamine and/or methamphetamine, said method comprising:
(a) providing in combination in a medium:
(i) said sample and
(ii) a reagent system according to claim 20 ; and
(b) examining said medium for the presence of a complex comprising said amphetamine and said antibody for amphetamine and/or a complex of said methamphetamine and said antibody for methamphetamine, the presence thereof indicating the presence of said amphetamine and/or methamphetamine in said sample.
32. A method according to claim 31 wherein said examining comprises measuring signal from said enzyme, the amount thereof being related to the presence of said amphetamine and/or methamphetamine in said sample.
33. A method according to claim 32 wherein said method is a homogeneous method and said medium is examined for the amount of said signal.
34. A method according to claim 32 wherein said method is a heterogeneous method and said complex, if present, is separated from said medium and said medium or said complex is examined for the amount of said signal.
35. A method for determining amphetamine and/or methamphetamine in a sample suspected of containing amphetamine and/or methamphetamine, said method comprising:
(a) providing in combination in a medium:
(i) said sample and
(ii) a reagent system according to claim 21 ; and
(b) examining said medium for the presence of a complex comprising said amphetamine and said antibody for amphetamine and/or a complex of said methamphetamine and said antibody for methamphetamine, the presence thereof indicating the presence of said amphetamine and/or methamphetamine in said sample.
36. A method according to claim 35 wherein said examining comprises measuring signal from said enzyme, the amount thereof being related to the presence of said amphetamine and/or methamphetamine in said sample.
37. A method according to claim 36 wherein said method is a homogeneous method and said medium is examined for the amount of said signal.
38. A method according to claim 36 wherein said method is a heterogeneous method and said complex, if present, is separated from said medium and said medium or said complex is examined for the amount of said signal.
39. A method for determining amphetamine and/or methamphetamine in a sample suspected of containing amphetamine and/or methamphetamine, said method comprising:
(a) providing in combination in a medium:
(i) said sample and
(ii) a reagent system according to claim 29 ; and
(b) examining said medium for the presence of a complex comprising said amphetamine and said antibody for amphetamine and/or a complex of said methamphetamine and said antibody for methamphetamine, the presence thereof indicating the presence of said amphetamine and/or methamphetamine in said sample.
40. A method according to claim 39 wherein said examining comprises measuring signal from said enzyme, the amount thereof being related to the presence of said amphetamine and/or methamphetamine in said sample.
41. A method according to claim 40 wherein said method is a homogeneous method and said medium is examined for the amount of said signal.
42. A method according to claim 40 wherein said method is a heterogeneous method and said complex, if present, is separated from said medium and said medium or said complex is examined for the amount of said signal.
43. A method for determining amphetamine and/or methamphetamine in a sample suspected of containing amphetamine and/or methamphetamine, said method comprising:
(a) providing in combination in a medium:
(i) said sample and
(ii) a reagent system according to claim 30 ; and
(b) examining said medium for the presence of a complex comprising said amphetamine and said antibody for amphetamine and/or a complex of said methamphetamine and said antibody for methamphetamine, the presence thereof indicating the presence of said amphetamine and/or methamphetamine in said sample.
44. A method according to claim 43 wherein said examining comprises measuring signal from said enzyme, the amount thereof being related to the presence of said amphetamine and/or methamphetamine in said sample.
45. A method according to claim 44 wherein said method is a homogeneous method and said medium is examined for the amount of said signal.
46. A method according to claim 44 wherein said method is a heterogeneous method and said complex, if present, is separated from said medium and said medium or said complex is examined for the amount of said signal.
47. A method for determining amphetamine and/or methamphetamine in a sample suspected of containing amphetamine and/or methamphetamine, said method comprising:
(a) providing in combination in a medium:
(i) said sample,
(ii) an antibody for amphetamine,
(iii) an antibody for methamphetamine,
(iv) a compound of the formula:
wherein:
R 1 ′ is H, lower alkyl, a protecting group,
R 2 ′ is H, lower alkyl, a protecting group,
W′ is O, S, or NR 3 ′ wherein R 3 ′ is H or lower alkyl,
X′ is C(O) or SO 2 ,
Y′ is bond, S or —NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Z′ is an enzyme,
t′ is an integer from 1 to 6 and v′ is an integer from 2 to 6,
n′ is an integer between 1 and the molecular weight of said enzyme divided by 500; and
(b) examining said medium for the presence of a complex comprising said amphetamine and said antibody for amphetamine and/or a complex of said methamphetamine and said antibody for methamphetamine, the presence thereof indicating the presence of said amphetamine and/or methamphetamine in said sample.
48. A method according to claim 47 wherein said examining comprises measuring signal from said enzyme, the amount thereof being related to the presence of said amphetamine and/or methamphetamine in said sample.
49. A method according to claim 48 wherein said method is a homogeneous method and said medium is examined for the amount of said signal.
50. A method according to claim 48 wherein said method is a heterogeneous method and said complex, if present, is separated from said medium and said medium or said complex is examined for the amount of said signal.
51. A method according to claim 47 wherein said enzyme is glucose-6-phosphate dehydrogenase.
52. A method for determining amphetamine and/or methamphetamine in a sample suspected of containing amphetamine and/or methamphetamine, said method comprising:
(a) providing in combination in a medium:
(i) said sample,
(ii) a conjugate of an enzyme and an amphetamine analog and/or a conjugate of an enzyme and a methamphetamine analog,
(iii) an antibody for amphetamine, said antibody being raised against a compound of the formula:
wherein:
R 1 ′ is H and R 2 ′ is H,
W′ is O, S, or NR 3 ′ wherein R 3 ′ is H or lower alkyl,
X′ is C(O) or SO 2 ,
Y′ is bond, S or —NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Z′ is a protein immunogenic carrier or a non-poly(amino acid) immunogenic carrier,
t′ is an integer from 1 to 6 and v′ is an integer from 2 to 6,
n′ is an integer between 1 and the molecular weight of said antigen or said immunogenic carrier divided by 500; and/or
(iv) an antibody for methamphetamine, said antibody being raised against a compound of the formula:
wherein:
R 1 ′ is H and R 2 ′ is methyl,
W′ is O, S, or NR 3 ′ wherein R 3 ′ is H or lower alkyl,
X′ is C(O) or SO 2 ,
Y′ is bond, S or —NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Z′ is a protein immunogenic carrier or a non-poly(amino acid) immunogenic carrier,
t′ is an integer from 1 to 6 and v′ is an integer from 2 to 6,
n′ is an integer between 1 and the molecular weight of said antigen or said immunogenic carrier divided by 500; and
(b) examining said medium for the presence of a complex comprising said amphetamine and said antibody for amphetamine and/or a complex of said methamphetamine and said antibody for methamphetamine, the presence thereof indicating the presence of said amphetamine and/or methamphetamine in said sample.
53. A method according to claim 52 wherein said examining comprises measuring signal from said enzyme, the amount thereof being related to the presence of said amphetamine and/or methamphetamine in said sample.
54. A method according to claim 53 wherein said method is a homogeneous method and said medium is examined for the amount of said signal.
55. A method according to claim 53 wherein said method is a heterogeneous method and said complex, if present, is separated from said medium and said medium or said complex is examined for the amount of said signal.
56. A method according to claim 52 wherein said enzyme is glucose-6-phosphate dehydrogenase.
57. A kit comprising in packaged combination:
(i) an antibody for amphetamine,
(ii) an antibody for methamphetamine,
(iii) a compound of the formula:
wherein:
R 1 ′ is H, lower alkyl, a protecting group,
R 2 ′ is H, lower alkyl, a protecting group,
W′ is O, S, or NR 3 ′ wherein R 3 ′ is H or lower alkyl,
X′ is C(O) or SO 2 ,
Y′ is bond, S or —NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Z′ is an enzyme,
t′ is an integer from 1 to 6 and v′ is an integer from 2 to 6,
n′ is an integer between 1 and the molecular weight of said enzyme divided by 500.
58. A kit according to claim 57 wherein said enzyme is glucose-6-phosphate dehydrogenase.
59. A kit comprising in packaged combination:
(i) a conjugate of an enzyme and an amphetamine analog and/or a conjugate of an enzyme and a methamphetamine analog,
(ii) an antibody for amphetamine, said antibody being raised against a compound of the formula:
wherein:
R 1 ′ is H and R 2 ′ is H,
W′ is O, S, or NR 3 ′ wherein R 3 ′ is H or lower alkyl,
X′ is C(O) or SO 2 ,
Y′ is bond, S or —NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Z′ is a protein immunogenic carrier or a non-poly(amino acid) immunogenic carrier,
t′ is an integer from 1 to 6 and v′ is an integer from 2 to 6,
n′ is an integer between 1 and the molecular weight of said antigen or said immunogenic carrier divided by 500; and/or
(iii) an antibody for methamphetamine, said antibody being raised against a compound of the formula:
wherein:
R 1 ′ is H and R 2 ′ is methyl,
W″ is O, S, or NR 3 ′ wherein R 3 ′ is H or lower alkyl,
X′ is C(O) or SO 2 ,
Y′ is bond, S or —NR 3 ′ wherein R 3 ′ is H or lower alkyl,
Z′ is a protein immunogenic carrier or a non-poly(amino acid) immunogenic carrier,
t′ is an integer from 1 to 6 and v′ is an integer from 2 to 6,
n′ is an integer between 1 and the molecular weight of said antigen or said immunogenic carrier divided by 500.