Fluorescent Imaging

Antibodies are indispensable tools for biomedical research applicable in fluorescent imaging of cells and tissue. The antibody conjugation method is a crucial step in order to obtain high-quality imaging results. GlyCLICK delivers site-specific and quantitative labeling for conjugates with preserved antigen binding capacity for reproducible results in sensitive imaging applications.

GlyCLICK is a versatile technology that facilitates the generation of tailored conjugates suitable for fluorescent imaging applications such as in vitro cell imaging, immunohistochemistry (IHC) and flow cytometry (FCM). The GlyCLICK kit is available with the AlexaFluor®488 label or for Azide Activation only that allows for the conjugation using custom cyclooctyne-functionalized labels of choice.

GlyCLICK workflow - Fluorescent Imaging

High-quality in vitro cell imaging

Labeling of antibodies with fluorescent dyes is commonly used for immunostaining of cells in imaging applications. Indirect detection methods are limited by the primary antibody species and often require low primary antibody concentration that may compromise the complete binding to cells. The site-specific GlyCLICK technology enables quantitative conjugation for the detection of cells and increased flexibility for multi-staining analysis in fluorescent imaging.

Confocal microscopy of cells with HER2 high (+) expression was performed using a direct detection method with GlyCLICK conjugated trastuzumab-AlexaFluor®647 and indirect detection using secondary labeled antibodies optimized for imaging applications (Fig. 1). The site-specific GlyCLICK conjugates provide distinct and dynamic cell membrane labeling for high-quality images. Direct detection using GlyCLICK conjugates also showed a high signal-to-noise ratio superior to that of indirect labeling.

Fluorescent Imaging GlyCLICK

Figure 1. HER2(+) cells fixated with PFA (2%) were incubated with a) GlyCLICK or b) randomly conjugated trastuzumab (1 µg/ml) for 1 hour. Cells were also incubated with c) mouse anti-human (3B5) primary antibody (0.2 µg/ml) for 1 hour and AlexaFluor488-conjugated donkey anti-mouse secondary antibody (1:200) for 30 minutes. Incubated cells were stained with DAPI (0.2 µM) for 2 hours and imaged using confocal microscopy. 

Quantitative conjugation for histological imaging

The quality and affinity of the conjugated antibody is crucial in IHC methods in order to obtain reliable and reproducible results. Since GlyCLICK generates conjugates with a degree of label that equals two (DOL=2.0) and preserves full antigen binding affinity, reliable and quantitative results in sensitive imaging applications can be achieved. The GlyCLICK kit was used for site-specific conjugation of trastuzumab-AlexaFluor®647 (T-GlyCLICK-AlexaFluor®647) and analyzed with LC-MS (Fig. 2a,b). Data was compared to a standard commercial kit for random labeling (Fig. 2c). GlyCLICK conjugates displayed a constant degree of label (DOL=2.0) compared to the heterogenous pool of conjugates generated by the random labeling approach.

Fluorescent imaging – GlyCLICK

Figure 2. Fc subunit analysis by LC-MS on GlyCLICK and randomly conjugated antibodies after digestion with FabRICATOR. Samples of a) trastuzumab Fc deglycosylated to the inner GlcNAc using GlycINATOR, b) T-GlyCLICK-AlexaFluor®647 conjugate,c) randomly conjugated trastuzumab-AlexaFluor®647. Fc subunits were separated from Fd and LC by reversed phase on a Protein BEH C4 column from Waters and analyzed on a Bruker Impact II ESI Q-TOF. Mass spectra were deconvoluted using the MaxEnt algorithm.

 

The labeled antibodies (Fig. 2) were used for imaging with confocal and wide-field epi-fluorescense microscopy in HER2(+) human breast tissue to assess the performance of the different conjugates (Fig. 3).  The GlyCLICK site-specific labeled antibodies allowed for detection with confocal and wide-field imaging of highly expressed antigens providing more dynamic images with good visualization of distinctly labeled membranes in the tissue sections.

Fluorescent imaging

Figure 3. Confocal (a, b) and wide-field epi-fluorescense microscopy (c, d) of paraffin embedded HER2(+) human breast tissue with trastuzumab-AlexaFlour®647 conjugates using GlyCLICK and random labelling (10 ug/ml). HIER processed tissue was blocked (BSA) and incubated with GlyCLICK or randomly conjugated antibodies (10 µg/ml) for 90 minutes prior to DAPI incubation (0.2 µM) and mounting in antifade solution. 

High-affinity binding for flow cytometry applications

Fluorescent antibodies are valuable tools in qualitative and quantitative analysis using Flow Cytometry (FCM). Indirect detection methods require several incubation steps and secondary antibodies are a potential source of unsolicited background staining due to unspecific binding or cross-reactivity.Site-specific and quantitative conjugation using GlyCLICK enables direct labeling of cells with a single incubation step and increased flexibility in primary antibody selection for multiplexing experiments.

The performance of directly labeled antibodies conjugated using GlyCLICK was evaluated by FCM (Fig. 4). Trastuzumab was labeled with AlexaFluor®647 (T-GlyCLICK-AlexaFluor®647) or Cy5 (T-GlyCLICK-Cy5) using the GlyCLICK technology for direct detection of HER2(+) cells and analyzed using FCM. An indirect detection method was also evaluated using a commercially available labeling method optimized for FCM analysis. The results show that the GlyCLICK conjugates provided a 10-fold increase in separation index (SI) due to the lower background signal, compared to the indirect detection method.

Figure 4. FCM analysis of HER2(+) cells (MDA-MB-435) treated with human Fc blocking mix (Miltenyi Biotech) and incubated with GlyCLICK conjugates or mouse primary antibody (3.7 ug/ml) for 30 minutes. Cells incubated with mouse primary antibody were incubated with secondary donkey anti-mouse antibody solution (1:200) for 30 minutes.Resuspended (PBS+ 1% BSA) cells were then analyzed using a CytoFlex flow cytometer. The analysis was performed by Truly Labs AB, Sweden.

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GlyCLICK® is a site-specific conjugation technology for IgG using enzymatic remodeling and click-chemistry.

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