Precolumn affinity capillary electrophoresis for the identification of clinically relevant proteins in human serum: Application to human cardiac troponin I

An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of c apillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, a t clinically relevant levels (2 mmol/L) in serum is demonstrated, The techn ique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (similar to 5 mm) packed bed of porous silica containing covalentl y immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex mat rix. Following a rinsing step to eliminate nonspecifically bound serum prot eins and other impurities from the column, desorption of the antigen into t he separation region of the PA-CE capillary for subsequent measurement of f emtomolar amounts of cTnI by CE is effected by the injection of an appropri ate elution buffer. Advantages of this approach over previously reported af finity preconcentration techniques, related applications for PA-CE technolo gy, and its potential for use in the development of a certified reference m aterial for cTnI in serum are discussed.