Sensitive Detection of Biomarkers using GlyCLICK®

Application

Higher-resolution flow cytometry with improved signal intensity and a 10-fold increase in separation index using site-specific antibody labeling.

Flow cytometry enables high-throughput cell sorting and multiplexed analysis by measuring multiple markers on individual cells in a heterogeneous population. While indirect detection using fluorescent antibodies is widely used, advanced conjugation technologies can provide more direct, consistent, and quantitative readouts, improving sensitivity and reducing variability in complex assays.

Multiplexing can result in unwanted fluorescence signals and noise levels originating from unspecific staining, elevated background and spillover that reduces both the sensitivity and resolution of the analysis. The ability to discriminate between negative and positive populations of cells can thus be affected, and a low separation index is often observed. The performance of site-specifically labeled antibodies conjugated using GlyCLICK was evaluated for flow cytometry imaging and compared to an indirect detection method.

GlyCLICK conjugation is a powerful alternative to indirect detection methods

HER2 positive cells were immunostained with T-GlyCLICK-AlexaFluor®647 or T-GlyCLICK-Cy5 and compared to indirect detection using primary and secondary antibodies. To minimize unspecific binding, titration was determined the saturation level of 3.7 ug/ml. The results show both higher intensities and a better separation of negative and positive populations with a 10-fold increase in separation index using GlyCLICK conjugated antibodies.

Improved signal intensities with GlyCLICK conjugates in flow cytometry applications

 Fluorescent imaging of high expressing HER2-positive cells. Titration curve of MDA-MB-453 cells against antibody in solution 1:200 (0.046-100 µg/ml) (left). Flow cytometry analysis showing fluorescence intensity and count of MDA-MB-453 cells either unstained with donkey anti-mouse-AF647 (control) or stained with mouse anti-HER2 + donkey anti-mouse-AF647, T-GlyCLICK-AlexaFluor®647 or T-GlyCLICK-Cy5 (right).

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