Abstract
Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. The high pH reduces protein adsorption but reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.
Highlights
Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders
Refractive index detection using BSI is combined with fluorescence measurements to develop an high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay quantification
SPE profiles in boric acid, arginine, and cyclohexyl-2-aminoethanesulfonic acid (CHES) buffers were measured with BSI and showed the characteristic five major protein bands expected for SPE
Summary
Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Electrophoretic separation of serum proteins was first demonstrated in the early 1900s by Tiselius using a U-shaped electrophoretic cell[5] This combination of moving boundary electrophoresis coupled with optical detection, marked the first separation of serum proteins into distinct protein bands[5]. Since these early studies, electrophoretic separations using cellulose acetate or more commonly agarose gels rapidly evolved and became the gold standard for SPE measurements[6]. The presence of antibiotics, for instance, has been shown to lead to unwanted contributions to the SPE s ignal[22]
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