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This study demonstrates an improved magnetic protein microsphere-aided sandwich fluoroimmunoassay for the analysis of myoglobin and heart-type fatty acid binding protein (H-FABP), early protein markers associated with acute myocardial infarction. In preparation for the assay we constructed superparamagnetic human serum albumin (HSA)/gamma-Fe(2)O(3) microspheres, and grafted capture antibodies (monoclonal antimyoglobin 7C3 and anti-H-FABP 10E1) onto the protein microspheres using the avidin-biotin system. Then the antibody-carrying microspheres were used in a sequential sandwich fluoroimmunoassay along with detection antibodies (Alexa fluor594-labeled antimyoglobin 4E2 and FITC-labeled anti-H-FABP 9F3). The magnetic HSA/gamma-Fe(2)O(3) microspheres were characterized by scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectrophotometry, atomic absorption spectrophotometry, and vibrating sample magnetometry. Fluorescence images of the post-immunoassay microspheres recorded using an inverted fluorescence microscope showed that the average fluorescence intensity was correlated with the concentration of cardiac markers, in agreement with the results obtained by an F-4500 FL spectrophotometer; this indicated that the fluoroimmunoassay could be used to semiquantitatively detect both myoglobin and H-FABP. The detection limit was 10 ng/mL for myoglobin and 1 ng/mL for H-FABP.

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