Methods for the preparation of antibodies directed against dithiocarbamates and the use thereof for detection of nitric oxide in body fluids
First Claim
1. A method for rendering a dithiocarbamate immunogenic, said method comprising contacting said dithiocarbamate with a macromolecule in the presence of a crosslinking agent under crosslinking conditions wherein said dithiocarbamate and said macromolecule are covalently attached via said crosslinking agent, rendering said dithiocarbamate immunogenic.
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Abstract
In accordance with the present invention, ELISA methods for the measurement of NO levels in mammalian body fluids utilizing monoclonal antibodies directed against dithiocarbamates and related iron complexes are described. It has been found that conjugation of dithiocarbamates to a macromolecule produces immunogenic dithiocarbamate-macromolecule derivatives. Such derivatives can be used for the production (e.g., in rodents) of monoclonal antibodies directed against different forms of dithiocarbamates (e.g., free dithiocarbamates, as well as complexes thereof with iron and, optionally, nitric oxide). In contrast, non-derivatized dithiocarbamates alone are not immunogenic. The simple, easy and non-invasive ELISA methods for measurement of NO levels in body fluids will find a variety of uses, e.g., for diagnosis and monitoring of NO overproduction that has been associated with many inflammatory and infectious diseases.
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Citations
18 Claims
- 1. A method for rendering a dithiocarbamate immunogenic, said method comprising contacting said dithiocarbamate with a macromolecule in the presence of a crosslinking agent under crosslinking conditions wherein said dithiocarbamate and said macromolecule are covalently attached via said crosslinking agent, rendering said dithiocarbamate immunogenic.
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14. A method for preparing an immunogenic dithiocarbamate from a non-immunogenic dithiocarbamate, said method comprising contacting said non-immunogenic dithiocarbamate with a macromolecule in the presence of a crosslinking agent under crosslinking conditions wherein said dithiocarbamate and said macromolecule are covalently attached via said crosslinking agent, rendering said dithiocarbamate immunogenic.
- 16. An immunogenic dithiocarbamate comprising at least one dithiocarbamate covalently attached via a crosslinking agent to a macromolecule, wherein said dithiocarbamate has the structure
- space="preserve" listing-type="equation">(R).sub.2 N--C(S)--SH,
wherein each R is independently selected from a C1 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 form a 5-, 6- or 7-membered ring including the N and the two R groups, and wherein said macromolecule is immunogenic when crosslinked with said dithiocarbamate and is not an antibody. - View Dependent Claims (17, 18)
Specification