Method of assaying pyrrole-containing biological compounds
View Patent ↗This invention relates to a method of assaying pyrrole-containing biological compounds and chemical compositions that can be used in the method. The method involves contacting a biological compound with one of: a) a bound or bindable derivatizing agent which forms a reaction product with the biological compound, followed by exposure to a detectable molecule which forms a complex with the reaction product; or b) a derivatizing agent which forms a reaction product with the biological compound, followed by exposure to a bound binding agent specific to the biological compound in the reaction product; or c) a binding agent specific to the biological compound, followed by exposure to a derivatizing agent which forms a reaction product with the biological compound, and determining the amount of bound biological compound. There is also provided a method of preparing an antigen.
1. A method for isolating pyrrole-containing biological compounds from a sample, the method comprising:
1) contacting the sample with a biotinylated Ehrlich's reagent wherein the biotinylated Ehrlich's reagent comprises a cleavable bond attached through the amine moiety of Ehrlich's reagent and the cleavable bond links the Ehrlich's reagent to biotin such that a reaction product is formed between the pyrrole-containing biological compound and the biotinylated Ehrlich's reagent;
2) contacting the reaction product with avidin or streptavidin attached to a solid support, such that the reaction product is bound to the solid support through biotin-avidin/streptavidin binding; and
3) isolating the reaction product bound to the support by cleaving the cleavable bond of the Ehrlich's reagent.
2. The method according to claim 1 , wherein the Ehrlich's reagent has a formula of
wherein R 1 is an alkyl group, R 2 is an alkylene group, R 4 is a heteroalkylene group comprising a cleavable bond, X is biotin, and Y is avidin or strepavidin attached to a solid support.
3. The method according to claim 1 , further comprising washing the reaction product bound to the solid support prior to the isolating step (3).
4. The method according to claim 2 , wherein R 1 is a straight-chain alkyl group containing 1–10 carbon atoms, and R 2 is a straight-chain alkylene group containing 1–10 carbon atoms.
5. The method according to claim 4 , wherein R 1 is a straight-chain alkyl group containing 1–5 carbon atoms.
6. The method according to claim 4 , R 2 is a straight-chain alkylene group containing 1–5 carbon atoms.
7. The method according to claim 6 , wherein R 1 is a straight-chain alkyl group containing 1–5 carbon atoms, and R 2 is a straight-chain alkylene group containing 1–5 carbon atoms.
8. The method according to claim 1 , wherein R 4 comprises a disulphide bond.
9. The method according to claim 8 , wherein R 4 is —NH(CH 2 ) 2 SS(CH 2 ) 2 NHC(O)(CH 2 ) 5 NH—.
10. The method according to claim 8 , wherein the disulphide bond is cleaved by a reducing agent.
11. The method according to claim 10 , wherein the reducing agent is β-Mercaptoethanol and dithiothreitol.
12. The method according to claim 10 , wherein the reducing agent is dithiothreitol.
13. A method for isolating pyrrole-containing biological compounds from a sample, the method comprising:
1) contacting the sample with a biotinylated Ehrlich's reagent which comprises a cleavable bond such that a reaction product is formed between the pyrrole-containing biological compound and the biotinylated Ehrlich's reagent; wherein the biotinylated Ehrlich's reagent has a formula of:
wherein R 1 is methyl, R 2 is ethylene, R 4 is —NH(CH 2 ) 2 SS(CH 2 ) 2 NHC(O)(CH 2 ) 5 NH— which comprises a disulphide bond, and X is biotin,
2) contacting the reaction product with avidin immobilized on a column, such that the reaction product is bound to the solid support through biotin-avidin binding; and
3) applying dithiothreitol to the column, thereby cleaving the disulphide and isolating the reaction product bound to the support.