Schiff-base conjugate of n, n-dibutyl-p-phenylenediamine with pyridoxal 5'-phosphate for improved homocysteine assays using pyridoxal 5'-phosphate-dependent enzymes

Abstract

A composition, method and kit for performing a two-reagent enzymatic homocysteine assay, wherein a single homocysteinase enzyme and a Schiff-based conjugate of N,N-dibutyl-p-phenyldiamine (DBPDA) with pyridoxal 5′-phosphate (PLP) are used to measure total homocysteine in plasma or serum. The assay measures a chromophore reaction product of H 2 S and the DBPDA released from the Schiff-base conjugate in the presence of a Fe +3 containing compound. The resulting chromophore may be measured absorbance or fluorescence spectrophotometry.

What is claimed is: 
     
         1 . A kit comprising:
 a first reagent, the first reagent including a mixture of a reducing agent, homocysteinase (HCYase) and N,N-dibutyl-p-phenyldiamine-pyridoxal 5′-phosphate;   a second reagent, the second reagent including an Fe(III) containing compound;   a first buffer solution; and   a second buffer solution.   
     
     
         2 . The kit of  claim 1 , wherein the HCYase is L-homocysteine α,γ-lyase. 
     
     
         3 . The kit of  claim 1 , wherein the Fe(III) containing compound is FeCl 3 . 
     
     
         4 . The kit of  claim 1 , wherein the first buffer solution is a sodium phosphate buffer. 
     
     
         5 . The kit of  claim 4 , wherein the first buffer solution further includes a non-ionic surfactant. 
     
     
         6 . The kit of  claim 4 , wherein the second buffer solution is a strong acid. 
     
     
         7 . The kit of  claim 5  wherein the non-ionic surfactant is triton X-100. 
     
     
         8 . The kit of  claim 1 , further comprising at least one calibrator and at least one control. 
     
     
         9 . The kit of claim  25 , wherein the at least one calibrator and the at least one control are each a matrix plasma containing total homocysteine of a known concentration.