Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers.
OBJECTIVE
The multiplexed, microsphere-based flow cytometric assay (MFCA) for multiple human tumor markers was established for the early screening and detection of suspected cancer patients.


METHODS
Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were identified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples.


RESULTS
The identifications revealed that the coupling procedures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality control of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional "gold standard", which was the ELISA.


CONCLUSIONS
The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications.
Sun
Kai
K
Department of Hepatobiliary Surgery, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China.
Wang
Qian
Q
Huang
Xiao-hui
XH
Zhen
Mao-chuan
MC
Li
Wen
W
Zhang
Long-juan
LJ
D014408:Biomarkers, Tumor / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D008863:Microspheres; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D014408:Biomarkers, Tumor