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Related Concept Videos

Flow Cytometry01:23

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy
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Quantifying multimodal contrast agent biological activity using near-infrared flow cytometry.

Mary A Hall1, Melissa B Aldrich, Ali Azhdarinia

  • 1Center for Molecular Imaging, The Brown Foundation Institute of Molecular Medicine at The University of Texas Health Science Center, Houston, TX 77030, USA.

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|April 28, 2012
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A new near-infrared (NIR) flow cytometry (FC) method accurately quantifies the biological activity of dual-labeled antibodies for multimodal imaging, offering greater reproducibility than the Lindmo assay.

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Area of Science:

  • Biomedical Engineering
  • Molecular Imaging
  • Biotechnology

Background:

  • Biological activity validation is crucial for imaging agents before clinical use.
  • The conventional Lindmo assay for quantifying radiolabeled antibody immunoreactivity may lack consistent accuracy.
  • Multimodal contrast agents combine positron emission tomography and NIR fluorescence imaging.

Purpose of the Study:

  • To develop and validate a near-infrared (NIR) flow cytometry (FC) method for quantifying the biological activity of dual-labeled antibodies.
  • To compare the accuracy and reproducibility of the NIR FC method with the conventional Lindmo assay.
  • To assess the impact of copper (Cu) labeling on the antibody's target-binding ability.

Main Methods:

  • Developed a NIR FC assay for dual-labeled antibodies targeting epithelial cell adhesion molecule.
  • Conjugated antibody with DOTA-NHS-ester, labeled with IRDye 800CW, and (64)Cu or nonradioactive Cu.
  • Tested agent's biological activity using both Lindmo assay and NIR FC with human prostate cancer cells.

Main Results:

  • Lindmo assay showed 76.4 ± 15.7% immunoreactivity.
  • NIR FC yielded 73.1 ± 7.7% (with Cu) and 79.4 ± 8.1% (without Cu) biological activity, with no significant difference (p > 0.05).
  • Increased NIR dye-to-protein ratio significantly reduced biological activity in both assays. Cu labeling did not significantly affect target binding.

Conclusions:

  • NIR FC is a reproducible and accurate alternative to the Lindmo assay for quantifying biological activity of NIR fluorophore-labeled multimodal imaging agents.
  • The developed NIR FC method demonstrates that Cu labeling does not compromise the antibody's specific binding.
  • This validates NIR FC for assessing the biological activity of advanced imaging agents.