Competitive Enzyme-linked Immunoassay For Determining Total Homocysteine In Plasma Or Serum

Objective: Many recent studies showed that an increased level of total homocysteine (tHcy) in plasma or serum is correlated with many diseases. Particularly, it has been considered as an independent risk factor of cardiocascular diseases. In this study, we try to establish a quantitive immunoassay for tHcy in plasma or serum.Methods: S-adenosyl-L-homocysteine-bovine serum albumin (SAH-BSA) conjugation was prepared by succinylation. Monoclonal antibody(mAb) to SAH was prepared by hybridoma technology, in which the parent cells included Sp2/0 cells and the spleen cells from female Balb/c mice immunized with SAH-BSAconjugate. The tHcy levels of 47 samples were measured by competitive ELISA and HPLC, respectively.Results: The SAH-BSA conjugate was successively prepared by succinylation and it molar ratio is about 4.47~5.70. The mAb prepared in this study is specific for SAH, without cross-reactivity with adenosine (Ado), cysteine (Cys) and, methionine (Met). But it did show a minor corss-reactivity with S-adenosyl-L-methionine. The competitive ELISA established in this study had a detectable range from 1 to 50 u mol/L. Its recovery and precision were 98%~101% and 1%~3%, respectively. The tHcy levels of the 47 samples measured by the competitive ELSA showed a good correlation with those measured by HPLC (r= 0.9863).