Abstract
A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers.
Highlights
Lung cancer is the second most common cancer and the leading cause of all cancer deaths worldwide[1]
We focused on the comparison of the results obtained in the preclinical validation of the QD-encoded microspheres (QDEM)-based
The immunodiagnostic complex consisting of a capture antibody, an antigen, and a detection antibody had to be assembled on the QDEM surface
Summary
Lung cancer is the second most common cancer and the leading cause of all cancer deaths worldwide[1]. A wide range of different bead-based microarrays have been developed and successfully applied to detection of specific cancer markers[12,13] and nucleic acids[14], as well as for multiplexed genotyping15,16,analysis of single nucleotide polymorphisms (SNPs)[17], allergy testing[18], identification of pathogens[19], and the detection of biological warfare agents[20].The detection principle is based on optical encoding and quantitative analysis of each analyte with an antigen-specific fluorophore-encoded microbead population carrying its unique optical code. BD Biosciences company have developed bead-based system for simultaneous detection up to 30 analytes, such as cytokines, chemokines, growth factors, immunoglobulins, and phosphorylated cell signaling proteins in serum, plasma, or tissue culture supernatant samples. Incorporation of QDs into microspheres allows obtaining a large amount of individual spectral codes providing an attractive alternative to the state-of-the-art suspension microarray diagnostic systems[26]
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