Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers

Abstract

Aim: 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. Conclusion: 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.

Development of a bead-based suspension array for the detection of pathogens in acute respiratory tract infections

“…14 Sputum DNA was prepared and quantified as previously described. 15,16 Viral nucleic acids were extracted using the MiniBEST Viral RNA/DNA Extraction Kit (Ver.5.0; TakaRa, Shiga, Japan) and were then reverse transcribed into cDNA with the TUREscipt 1st Strand cDNA Synthesis Kit (Aidlab Biotechnologies Co., Beijing, China). Supplementary Table 1 shows the limits of detection for each pathogen based on serial dilution.…”

Mechanisms of human complement factor B induction in sepsis and inhibition by activated protein C

“…Cells were cultured for 4 days and then stimulated with LPS or TNF-␣ and IFN-␥ for 48 h. Cell supernatant (50 l) was used to quantify hBf protein expression using a microsphere-based flow cytometric assay developed in house. A monoclonal anti-Bf antibody (1379, a gift from Dr. Joshua Thurman, University of Colorado) was used as a capture antibody by coupling it to region 80 x-MAP beads (Luminex, Austin, TX) as previously described (67). Briefly, 1.25 ϫ 10 5 beads were activated for 20 min in 80 l of activation buffer (0.1 M NaH2PO4 at pH 6.2) containing 10 l each of sulfo-NHS and EDC (each prepared fresh in ddH 2O at 50 mg/ml).…”

Early diagnosis of disease using microbead array technology: A review

“…Microsphere-based flow cytometry assays (MFCA) established by Luminex involves a capture antibody coupled covalently to internally dyed polystyrene microspheres. The fluorophores which are red and orange can identify presence of tumor markers, a reporter molecule labeled with a fluorescent marker binds to analyte captured on the bead for detection [106].…”

“…Table 2 highlights the comparison between reproducibility, upper and lower detection limits, simultaneous multi-analyte detection, sensitivity and accuracy of 5 tumor markers. The results showed that their multiplexed assay was reproducible, sensitive, accurate and compared well with ELISA as a standard assay [106].…”