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Introduction
Nanobodies (Nbs) are the smallest functional antigen-binding antibody fragments with excellent properties as tracers for diagnostic purposes. One of the limiting factors for their in vivo use is their reabsorption by the kidneys, reducing the imaging quality in the vicinity of the kidneys. Previous studies have shown the influence of the label on the kidney retention of Nbs. To better understand their re-uptake mechanisms, we aim to localize Nbs labeled with different fluorescent dyes, Alexa FluorTM 647 (AF647) and Sulfo-Cy5 (Cy5), within the kidneys on sub-organ and cellular level.

Methods
The HER2 Nb 2Rs15d was either labeled with AF647 or Cy5 via amine-reactive chemistry. The Nbs were then injected in naive mice and 1 h later, the mice were perfused with 1x PBS, 4% PFA and CUBIC-P (TCI chemicals). The kidneys were then excised and fixed in 4% PFA. One kidney was cleared using a delipidation solution and a refractive index matching solution: CUBIC-L and CUBIC-R (TCI chemicals). Immunohistochemistry (IHC) staining was performed on paraffin sections of the other kidney, incubated first with a rat anti-LAMP1 (1:100, IgG2a; Thermofisher 1D4B) then for 1 h incubation with a secondary anti-rat-antibody Alexa FluorTM 488 (1:500, IgG2a; Jackson) and Hoechst 33342 (1:200, Sigma B2261). Images were acquired using a LaVision Ultramicroscope II and a Axio scan Z.1.

Results/Discussion
3D images obtained with light sheet fluorescence microscopy (LSFM) indicated that 1 h after injection, the anti-HER2 Nb labeled with AF647 was located in the cortex while the Cy5-labeled Nb was present in the outer medulla of the kidney. 3D volumetric imaging allowed to recognize the different shape of the proximal tubules indicating that both dyes were reabsorbed in different sections of the proximal tubule (Figure 1). The IHC staining on the kidney sections confirmed the results obtained with the LSFM and located the Nb signal mainly on the brush border membrane of the proximal tubule cells. A higher magnification allowed to co-localize intracellular Nb-signal with lysosomes (LAMP1) (Figure 2).

Conclusion
These results indicate that the processing of fluorescently labeled Nbs after glomerular filtration differs according to the dye. This implies the involvement of distinct re-uptake mechanisms and metabolization pathways, contingent upon the segments of the proximal tubules.
Originele taal-2English
StatusUnpublished - 2024
EvenementEMIM 2024 - Porto, Portugal
Duur: 12 mrt 202415 mrt 2024

Conference

ConferenceEMIM 2024
Land/RegioPortugal
StadPorto
Periode12/03/2415/03/24

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