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1. WO1997043645 - ANTICORPS DIRIGES CONTRE DES DITHIOCARBAMATES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

That which is claimed is:

1. A method for rendering a low molecular weight dithiocarbamate immunogenic, said method comprising contacting said dithiocarbamate with a macromolecule in the presence of a crosslinking agent under crosslinking conditions.

2. A method according to claim 1 wherein said dithiocarbamate is selected from compounds having the structure:
(R)2N-C(S)-SH,
wherein each R is independently selected from a C1 up to C18 alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl, aroyl, substituted aroyl, acyl, or substituted acyl, or the two R groups can cooperate to form a 5-, 6- or 7-membered ring including N and the two R groups.

3. A method according to claim 1 wherein said macromolecule is a polypeptide, polysaccharide, or polynucleic acid.

4. A method according to claim 3 wherein said polypeptide is a synthetic, naturally occurring or modified naturally occurring protein.

5. A method according to claim 4 wherein said protein is selected from serum albumin, hemocyanin, ovalbumin or PPD.

6. A method according to claim 1 wherein said dithiocarbamate is crosslinked to said macromolecule by said crosslinking agent via any functionality on said macromolecule .

7. A method according to claim 6 wherein said functionality on said macromolecule is an amino, hydroxy, sulfhydryl, or carboxyl group.

8. A method according to claim 1 wherein said crosslinking agent is a photoreactive crossl inker or a bifunctional crosslinker.

9. A method according to claim 8 wherein said photoreactive crosslinker is an azido compound or a diazo compound .

10. A method according to claim 9 wherein said photoreactive crosslinker is selected from sulfo succinimidyl (4-azidosalicyl amido) hexanoate, azidobenzoyl hydrazide, N-5-azido-2-n i t r ob e n z oy 1 oxy s u c c i n i m i d e , N - 4 - ( p - a z i d o -sal icyl amido) butyl-3 ' (2 '-pyridyldithio) propionamide, p-azidophenylglyoxal monohydrate, 4-(p-azidosalicylamido) 4- ( iodoacetamido) butane , bis [ (/3-4-azidosalicylamido) ethyl ] disufide, N-hydroxysuccinimidyl 4-azidobenzoate, N-hydroxysul f osuccinimidy 1 4-azidobenzoate,

N-hydroxysuccinimidyl-4-azidosalicylic acid,

N-hydroxysul f osuccinimidy 1 -4-azidosalicyl ic acid, p-nitrophenyl-2-diazo-3 , 3 , 3-trifluoropropionate, 2-diazo-3 , 3 , 3-trifluorpropionyl chloride, N-succinimidyl- (4-azidophenyl) -1,3' -dithiopropionate, sulfosuccinimidyl( 4 -azidophenyldithio) propionate, sulfosuccinimidyl-2-( 7 -azido- 4 -me thy 1 coumarin- 3 -acetamide) ethyl-1 , 3 ' -dithiopropionate , sul f osuccinimidy 1 -7 -az ido-4 -methy lcoumar in- 3 -acetate , or sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) -ethyl-1,3' -dithiopropionate .

11. A method according to claim 8 wherein said bifunctional crosslinker is a homobifunctional crosslinker or a heterobifunctional crosslinker.

12. A method according to claim 11 wherein said homo functional crosslinker is selected from dimethyl adipimidate, dimethyl suberimidate, dimethyl pimilimidate, disuccinimidyl glutarate, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, bis(diazo-benzidine) , ethylene glycobis(succinimidylsuccinate) , disuccinimidyl tartrate, disulfosuccinimidyl tartrate, bismaleidohexane, glutaraldehyde, dithiobis (succinimidyl propionate), dithiobis (sulfosuccinimidyl propionate) , 1 , 4-di [ 3 ', 2 ' -pyridyldithio (propionamido) butane], N,N'-dicyclohexylcarbodiimide, bis[2-(succinimidyloxy-carbonyloxy) ethyl ] sulfone or dimethyl

3,3' -dithiobispropion-imidate .

13. A method according to claim 11 wherein said heterobifunctional crosslinker is selected from succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimidyl-4- (p-maleimidophenyl)butyrate,N- (γ-maleimido-butyryloxy) succinimide ester, N-succinimidyl (4-iodoacetyl) aminobenzoate , 4-succinimidyl oxycarbonyl-α- (2- pyridyldithio) toluene, sulfosuccinimidyl-6- [α-methyl-α-( 2 -pyridyldithio) toluamido] hexanoate, N-succinimidyl-3- ( 2-pyr idy ldithio) propionate,

3 -( 2 -pyridyl d i thio ) propionyl hydrazide, l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 3-(p-azidosalicylamido) butylamine, l,5-difluoro-2,4-dinitrobenzene or N-hydroxysuccinimidyl 2,3-dibromo-propionate.

14. A method for preparing immunogenic species from non-immunogenic dithiocarbamates, said method comprising contacting said dithiocarbamate with a macromolecule in the presence of a crosslinking agent under crosslinking conditions.

15. The product produced by the method of claim 1.

16. An immunogenic derivative of a dithiocarbamate comprising at least one dithiocarbamate covalently attached to a macromolecule.

17. An immunogenic derivative according to claim

16 wherein said derivative is complexed with iron.

18. An immunogenic derivative according to claim

17 wherein said derivative is further complexed with nitric oxide.

19. A method for the production of antibodies against a dithiocarbamate, said method comprising immunizing a host animal with the modified dithiocarbamate of claim 16.

20. A method for the production of antibodies against a dithiocarbamate, said method comprising immunizing a host animal with the modified dithiocarbamate of claim 17.

21. A method for the production of antibodies against a dithiocarbamate, said method comprising immunizing a host animal with the modified dithiocarbamate of claim 18.

22. An antibody produced by the method of claim 19.

23. An antibody produced by the method of claim 20 .

24. An antibody produced by the method of claim 21 .

25. An antibody which specifically binds dithiocarbamate.

26. An antibody according to claim 25 wherein said antibody is selective for free dithiocarbamate.

27. An antibody according to claim 25 wherein said antibody is selective for the iron-dithiocarbamate complex, [ (dithiocarbamate) 2Fe] .

28. An antibody according to claim 25 wherein said antibody is selective for the nitric oxide-iron-dithiocarbamate complex, [ (dithiocarbamate) 2Fe-NO] .

29. A method for determining NO levels in mammalian body fluids, said method comprising:
contacting an aliquot of mammalian body fluid with:
an iron-dithiocarbamate complex,
an antibody according to claim 28, and
a labeled antibody selective for free
dithiocarbamate or iron-dithiocarbamate
complex, and thereafter
determining the quantity of nitric oxide-containing, iron-containing dithiocarbamate complex, thereby indicating the level of NO in said body fluid.

30. A method according to claim 29 wherein said contacting is carried out in vitro.

31. A method according to claim 29 wherein said contacting is carried out in vivo.

32. A method according to claim 29 wherein said body fluid is selected from saliva, blood, synovial fluid, urine, or tears.

33. A method to determine the tissue distribution of NO-scavenging dithiocarbamates and iron-containing complexes thereof, said method comprising:
contacting a tissue sample or extract therefrom with an antibody according to claim 24, and thereafter
determining which samples form antigen-antibody complex in combination with said antibody.