Homogeneous, Rapid Luminescent Oxygen Channeling Immunoassay (LOCITM) for Homocysteine

Homocysteine (Hcy) is present in plasma primarily bound as disulfides with itself, Cys, and albumin (∼70%) (1)(2)(3)(4). Total homocysteine (tHcy) in serum or plasma is markedly increased in patients with cobalamin or folate deficiency (3), and decreases only when they are treated with the deficient vitamin. tHcy is therefore of clinical relevance, with reference values in fasting subjects of ∼5–15 μmol/L (1). In addition, even a moderate increase of Hcy (hyperhomocysteinemia) is a risk factor for premature cardiovascular disease (4). These disorders justify introduction of the tHcy assay in the routine clinical chemistry laboratory. The development of a rapid, homogeneous assay for Hcy in serum or plasma using the luminescent oxygen channeling immunoassay (LOCITM) (5) is described. An ELISA for the determination of tHcy based on the modification of sample tHcy by alkylation and detection of the alkylated product was described previously (6). The current assay is designed for tHcy determination in serum or plasma at clinically relevant concentrations. Chemical reactions involved in the assay can be divided into three steps: The first step involves the release of bound Hcy by reduction of serum disulfides with tris-(2-carboxyethyl)phosphine (TCEP); the second step involves the derivatization of Hcy and cysteine with CABA …