(337p) Fluorescent Polymerization-Based Signal Amplification of Antigenic Binding Events for Immunofluorescent Imaging of Cells

Immunofluorescent imaging of cells is a widely used and critical tool for researchers and clinical pathologists to assess both protein expression and localization within cells. Applications have included detection of cancer biomarkers, study of protein localization during organogenesis, and imaging of cell surface proteins associated with drug resistance and cell signaling. Though several antigens are readily detected using fluorescently labeled antibodies, numerous lower abundance antigens are only detectable with improved microscopic techniques or signal amplification methods that increase the number of fluorophores coupled to each antigen/antibody binding event. Fluorescent polymerization-based amplification (FPBA) is presented as a signal amplification platform suitable for immunocytochemistry that enables highly sensitive fluorescent detection of cellular antigens, with advantages over many currently employed fluorescent signal amplification methods.

Previous reports have highlighted the use of FPBA for sensitive detection of DNA and proteins in a microarray format.^1,2 Here, we demonstrate that FPBA is a valuable method for immunofluorescent detection in cells. To achieve highly sensitive detection, FPBA takes advantage of the amplification inherent in radical chain polymerization. Specifically, photoinitiator molecules are coupled to protein probes, serving as a sensitive reporter for antigenic biodetection by initiating radical chain polymerization in the presence of monomer, light, and a fluorescent moiety, thereby forming a highly fluorescent gel specifically where an antigenic probe has bound its protein target. The work presented here employs a streptavidin-eosin conjugate (SA-eosin)³ that couples the photoinitiation capacity of eosin to the biorecognition function of streptavidin, a protein with an extremely high affinity for biotin, a common label for biological probes. SA-eosin specifically binds biotin-labeled antibody probes, thereby localizing eosin photoinitiators to regions of the surface where the biotin-labeled antibody probes have coupled to their antigenic target. A monomer formulation comprising 420 mM poly(ethylene glycol) diacrylate, 35 mM vinyl pyrrolidone, 210 mM N-methyldiethanolamine as a coinitiator, and 0.05 wt% nile red fluorescent NPs in water is applied to the surface which is subsequently exposed for 20 minutes to 30 mW/cm², > 480 nm light, generating a highly fluorescent film only where antibody/antigen recognition has occurred.

To assess the sensitivity of FPBA for immunofluorescent imaging of cells, FPBA staining was performed with decreasing concentrations of primary antibody against von Willebrand Factor (vWF), a protein that is abundant in the cytoplasm of lung endothelial cells, but is absent from fibroblast cells. Although for standard indirect immunofluorescence detection this antibody is recommended for use at 1:200 dilution, with FPBA, we are able to achieve clear visual distinction between positive staining in endothelial cells and background staining in fibroblast cells down to at least a 1:50,000 dilution of primary antibody, constituting an estimated 250-fold improvement in sensitivity. Also, in the absence of primary antibody, the endothelial cells do not show significant staining under these conditions, further verifying that the observed staining is specific for vWF. Based on the extremely low antibody concentrations that yield suitable vWF staining with FPBA, it is expected that FPBA will be capable of detecting lower abundance antigens that are difficult to detect by other approaches.

Since FPBA achieves signal amplification by generating a fluorescent polymer film with some inherent thickness, the suitability of FPBA for staining smaller, more localized cellular structures was investigated. Using primary antibody against the nuclear pore complex (NPC) proteins, FPBA was able to achieve distinct and specific staining of the nuclear membrane of human lung endothelial cells. The NPC is estimated to have a length of approximately 80 nm spanning through the nuclear envelope, while the estimated thickness of the observed fluorescent stain is approximately a few microns. Although the observed polymer film is much thicker than the actual size of the protein, the nuclear membrane is indeed visualized as a clearly distinguishable cellular structure. Further, in many applications, a stain that increases the feature thickness may be desirable as it will render the feature more readily observable. However, in situations in which a thinner film is required, it is likely that shorter polymerization times may be employed to reduce film thickness, while maintaining a bright fluorescent stain.

In conclusion, FPBA is found to be a highly sensitive signal amplification detection platform for immunofluorescent imaging of cells. Unlike other signal amplification strategies for cellular applications, FPBA does not employ peroxidase enzymes, and therefore is not hindered by the endogenous peroxidase activity in cells. In addition to microarrays and immunocytochemistry, it is expected that FPBA will also be advantageous for immunohistochemical imaging in tissues and flow cytometry-based cellular imaging techniques.

References

1. Avens, H. J., Bowman C. N. Acta Biomater, submitted.

2. Hansen, R. R.; Johnson, L. M.; Bowman, C. N. Anal Biochem 2009, 386, 285-287.

3. Hansen, R. R.; Sikes, H. D.; Bowman, C. N. Biomacromolecules 2008, 9, 355-362.