Cyclophosphamide analogs for use as immunogens and assay conjugates for an immunoassay of cyclophosphamide and ifosfamide
The present application relates to antibodies that bind to small molecules such as cyclophosphamide, ifosfamide, and analogs thereof, and immunological assays for determining the presence and/or quantifying the amount of cyclophosphamide and/or ifosfamide in a sample. By way of example, such immunological assays can be used for environmental testing.
1. A compound comprising a structure of Formula (II):
wherein
R 1b is —(CH 2 ) m —X, and one or more methylene units (—CH 2 —) is optionally replaced by oxygen or sulfur;
each R 2b and R 2b′ is independently selected from is —(CH 2 ) k —Z;
each R 3b , R 4b and R 5b is independently hydrogen, halogen, hydroxy, amino, cyano, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 7 cycloalkyl, 5-10 membered heterocyclyl, aryl, aralkyl, 5-10 membered heteroaryl, C 1 -C 6 alkoxy, C 1 -C 6 alkoxy(C 1 -C 6 )alkyl, aryloxy, halo(C 1 -C 6 )alkyl, halo(C 1 -C 6 )alkoxy, C 1 -C 6 alkylthio, arylthio, amino(C 1 -C 6 )alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, and oxo (═O);
X is a functional group selected from amine, thiol, carboxylic acid, hydrazide, hydrazine, oxime, or hydroxylamine;
Z is halogen or hydroxy; and
each m and k is independently an integer from 2 to 10; and
wherein the compound is covalently bonded to a polymer or carrier through the functional group X of the compound.
2. The compound of claim 1 , wherein Z is halogen.
3. The compound of claim 2 , wherein Z is fluoro or chloro.
4. The compound of claim 3 , wherein k is an integer of 2.
5. The compound of claim 4 , wherein each R 3b , R 4b and R 5b is hydrogen.
6. The compound of claim 1 , further comprising a carrier covalently bound to X, wherein the carrier comprises at least one of an albumin or fragment thereof, a serum protein, a lipoprotein, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovine thyroglobulin (BTG), a synthetic poly(amino acids), a poly amino-polysaccharide, a poly nucleic acid, a poly amino acid, a polysaccharide, or a solid particle.
7. The compound of claim 6 , wherein X is carboxylic acid.
8. The compound of claim 7 , wherein one or more methylene units (—CH 2 —) of R 1b is replaced by oxygen.
9. The compound of claim 1 , wherein k is 2, Z is chloro, m is 3, and X is carboxylic acid covalently linked to KLH.
10. The compound of claim 1 , wherein k is 2, wherein one or more methylene units (—CH 2 —) of R 1b is replaced by oxygen, and X is carboxylic acid covalently linked to BSA.
11. The compound of claim 1 , wherein the polymer or carrier is immunogenic.
12. The compound of claim 1 , wherein the immunogenic polymer or carrier is selected from an immunologically active protein or polypeptide.
13. The compound of claim 12 , wherein Z is halogen and k is 2.
14. The compound of claim 13 , wherein Z is fluoro or chloro.
15. The compound of claim 11 , wherein k is 2, Z is chloro, m is 3, and X is carboxylic acid covalently linked to KLH.
16. A kit for detecting the presence of cyclophosphamide and/or ifosfamide in a sample comprising reagents, one of the reagents being a conjugate of a carrier covalently bonded to a compound of claim 1 through the functional group X of the compound.
17. The kit of claim 16 , wherein k is 2, wherein one or more methylene units (—CH 2 —) of R 1b is replaced by oxygen, and X is carboxylic acid covalently linked to BSA.
18. A method of making an antibody, the method comprising:
administering the compound of claim 11 to a host organism;
isolating antibody-producing cells or antibodies from the host organism; and
screening antibodies derived from the antibody-producing cells or the antibodies from the host organism for affinity to cyclophosphamide.
19. The method of claim 18 , wherein k is 2, Z is chloro, m is 3, and X is carboxylic acid covalently linked to KLH.
20. The method of claim 18 , wherein the antibody-producing cells are isolated, and wherein monoclonal antibodies derived from the antibody-producing cells are screened for affinity to cyclophosphamide.
21. The method of claim 18 , further comprising screening for antibodies that bind specifically to cyclophosphamide compared to an acyclic metabolite of formula (III):
22. The method of claim 18 , wherein the antibodies from the host organism are screened for affinity to cyclophosphamide, and wherein the antibodies are polyclonal.
23. A method of making an antibody, the method comprising:
administering the compound of claim 11 to a host organism;
isolating antibody-producing cells or antibodies from the host organism; and
screening antibodies derived from the antibody-producing cells or the antibodies from the host organism for affinity to ifosfamide.
24. The method of claim 23 , wherein the antibody-producing cells are isolated, and wherein monoclonal antibodies derived from the antibody-producing cells are screened for affinity to ifosfamide.
25. The method of claim 23 , further comprising screening for antibodies that bind specifically to ifosfamide compared to an acyclic metabolite of formula (III):
26. The method of claim 23 , wherein the antibodies from the host organism are screened for affinity to ifosfamide, and wherein the antibodies are polyclonal.