CYCLICAL EXPONENTIAL AMPLIFICATION

We have developed a highly amplified and controllable immunofluorescence  method for use anywhere antibodies are used.  Our method generates signals well beyond what is possible with fluorescent secondary antibodies, and is useful for low-abundance antigens, high-background tissues, or anywhere brighter signals are needed.

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SEQUENTIAL BUILD UP OF FLUORESCENT, BRANCHED DNA

• Primary antibodies are tagged with a unique single-stranded DNA sequence

• A single stranded antisense detects the tag, forming a sticky end

• A branched T oligo finds that sticky end

• In turn, linear (L) fluorescent oligos binds the T

• T and L oligos are cyclically reapplied to build a DNA tree that is super bright,  stable, and removable.

• Sequences are biorthogonal for low-background.

 
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EXPONENTIAL AMPLIFICATION

•Signal intensity grows exponentially with cycle number.
•Can achieve much brighter signals than conventional fluorescent secondary antibodies.
•Stable after amplification.

 
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CONTROLLABLE AMPLIFICATION

The brightness can be controlled independently for each channel  by the:

•Number of amplification cycles.

•Number of fluorophores per oligo.

•The number of branches (3-way, 4-way, or higher).

Detection modality modifiable

•Large panel of fluorophores

•Haptens instead of fluorophores (biotin, digoxigenin…)

•Enzymes instead of fluorophores

 
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MULTIPLEX STAINING

•Distinct Oligo sets for each fluorescent channel, amplified simultaneously.
•Channel number only limited by the imaging system.
•A modular system: Any primary antibody can be linked to any fluorescence channel.
•Primary antibodies are detected by their tag, not their species, so any number of mouse, rabbit or other antibodies can be combined.
•Fluorescent DNA can be specifically and gently removed without tissue damage, allowing high-multiplex staining through multiple rounds of amplification and imaging.

 
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WORKFLOW

•Apply all antibodies at once.
•For a four-channel imaging system, use one for nuclei (DAPI), and three for antibodies
•Image on a standard fluorescence or multispectral imaging microscope.
•Strip the first three channels and amplify the next set of antibodies. Run as many rounds as needed to detect all antibodies.
•All steps run at room temperature and fit into typical IHC lab workflows.
•No specialized equipment needed.
•Compatible with autostainers and laboratory automation.

 

6-PLEX STAINING

Human Tonsil

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ROUND 1

CD68, PDL1, PD1

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ROUND 2

pan-Cytokeratin, CD8, CD3

 
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A BROADLY APPLICABLE, HIGHLY ADAPTABLE SYSTEM

Primary or secondary antibodies can be DNA-tagged
Projected  applications include:
•Immunohistochemistry in FFPE or frozen tissue
•Immunocytochemistry in cell lines
•Flow cytometry
•Western blots
•Bead assays
•ELISA
•In situ hybridization

 
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DNA-AMPLIFICATION IMPROVES LUMINEX BEAD ASSAY SENSITIVITY MORE THAN 10-FOLD

DNA-amplification detects Interleukin 6 at more than 10x lower concentration than with conventional phycoerythrin fluorescence.


Beads Imaged on a Luminex FLexMap 3D

 

MORE EXAMPLES

Human Tonsil

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Tumor

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Brain, FFPE

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Brain, ultrathin resin

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