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| Chemical name: | Cy5.5-Conjugated anti-human CD31 monoclonal antibody |
 |
| Abbreviated name: | Cy5.5-anti-hCD31 MAb | |
| Synonym: | ||
| Agent Category: | Antibody | |
| Target: | CD31 | |
| Target Category: | Binding | |
| Method of detection: | Fluorescence imaging | |
| Source of signal / contrast: | Cy5.5 | |
| Activation: | No | |
| Studies: |
| Structure of Cy5.5-anti-hCD31 MAb. |
[PubMed]
The CD31 molecule, also known as the platelet endothelial cell (EC) adhesion molecule 1, is a 130-kD transmembrane glycoprotein that is a member of the immunoglobulin superfamily. This glycoprotein is expressed primarily on the EC intercellular junctions in the vascular system, but it is also expressed to a varying extent on other cell types (1, 2). The ECs are believed to play a major role in a variety of physiological processes such as coagulation, vascular tone, inflammation, and angiogenesis (1). Although CD31 is expressed in several leukocyte cell subtypes and in platelets, it is considered to be a reliable marker for tumor vasculature (3, 4). Runnels et al. showed that fluorescence-labeled rat anti-mouse CD31 monoclonal antibodies (MAbs) targeting the CD31 molecule could be used for the in vivo imaging of EC dynamics in mice (5). The investigators showed that the MAbs could stain the ECs in the vascular wall for a period of 24 to 48 h after administration. On the basis of these observations, Bogdanov et al. designed a similar study using the anti-human CD31 MAb labeled with the near-infrared fluorescent dye Cy5.5 (Cy5.5 anti-hCD31) to investigate the detection of human EC-lined blood vessels developed in an animal model using human umbilical vein EC (HUVEC) Matrigel implants (6). To perform this study the investigators assumed that the human anti-CD31 MAb did not cross-react with the mouse ECs and had a high affinity for the targeted marker protein, that the CD31 was constitutively expressed on the surface of ECs, and that the MAb was internalized at a slow rate.
[PubMed]
The anti-human CD31 MAb used in the studies was received as a gift from a commercial source (6). The MAb, in 0.1 M sodium bicarbonate buffer (pH 8.7), was mixed with the N-hydroxysuccinimide monocarboxylic (NHS) ester of Cy5.5 (Cy5.5-NHS) in dimethylsulfoxide (in a dye:MAb molar ratio of 10:1). The mixture was incubated for 30 min (temperature not reported), and the labeled protein was purified with spin-chromatography on a mini spin column. A similar procedure was used to respectively conjugate a mouse anti-human E-selectin MAb fragment F(ab’)2 and a rat anti-mouse CD31 MAb with Alexa Fluor 488-NHS ester. Purification of these labeled MAbs was performed as described above. Ulex europaeus or Lycopersicon esculentum lectins were also respectively conjugated with Cy5.5-NHS and processed as given above. The purity and storage conditions used for the various conjugated MAbs and lectins were not reported.
[PubMed]
To test the fluorescent labeling of the two lectins, which have specificity for acetylglucosamine (L. esculentum) and α-L-fucose (U. europaeus), HUVEC and mouse vascular ECs (MVEC) were grown on glass-coverslip chambers and double-stained with the Cy5.5-labeled lectins and the anti-human or anti-mouse CD31 MABs (6). Both lectins stained the respective cells (HUVEC with the U. europaeus lectin and the MVEC with the L. esculentum lectin) as expected; however, the lectins did not show any specificity because the labeled lectins showed a cross-reactivity for both cell types. The species-specific anti-CD31 antibodies showed a homogeneous staining of the cells, indicating that the primary ECs were pure and did not contain any contaminating cells that would result in cross-staining with the labeled lectins. No competing studies with the unlabeled lectins or with the MAbs were reported.
In another study, the HUVEC cells were double-stained with the Cy5.5-anti-human CD31 MAb and the Alexa Fluor 488–conjugated anti-E-selectin MAb fragment F(ab’)2. Near-infrared fluorescent confocal microscope imaging revealed that the anti-human CD31 MAb (red fluorescence) was associated primarily with the intracellular junctions of the cells and that the E-selectin MAb (green fluorescence) showed little binding to the cells. Treatment of the cells with interleukin-1β (a pro-inflammatory cytokine; E-selectin has a role in inflammation) resulted in rapid cellular internalization of the E-selectin–labeled MAb, but there was only a little internalization of the anti-human CD31-labeled MAb under these conditions. Pre-exposure of the cells to N-ethylmaleimide or incubation (total incubation time not reported) at 4°C was reported to completely block internalization of either labeled MAb.
[PubMed]
Matrigel supplemented with growth factors (fibroblast growth factor and vascular endothelial growth factor) and mixed with or without HUVEC (to serve as controls) was injected into the right and left posterior flanks, respectively, of nu/nu mice to establish human cell-lined blood vessels in the animals (6). After approximately 3 to 4 weeks, the animals (n = 3 animals/group) were intravenously injected with the Cy5.5-anti-human CD31 MAb. Near-infrared imaging of the animals immediately after administration of the labeled MAb showed that the mouse body fluorescence was increased during the initial 5 min, followed by a gradual decrease as the imaging probe cleared from circulation. Approximately 2.5 h after injection, animals injected with the HUVEC Matrigel showed an increased fluorescence with a signal/noise ratio of 1.6–1.8 compared to the control animals. A histological examination with near-infrared fluorescence microscopy of frozen sections obtained from animals injected with Matrigel with or without HUVECs showed that the endothelium-lined blood vessels were visible only in animals with HUVEC Matrigel. No such structures were visible in frozen sections from the control animals.
The investigators triple-stained the HUVEC Matrigel implants with the Cy5.5-anti-human CD31 MAb, the Alexa Fluor 488–anti-mouse CD31 MAb, and 4,6'-diamidino-2-phenylindole (DAPI; blue fluorescent stain for nuclei) (6). A network of blood vessels almost entirely positive for the anti-human CD31 MAb and DAPI was observed in the HUVEC Matrigel sections. No staining with the labeled anti-mouse CD31 MAb was observed in these sections.
Some studies presented in this chapter were funded by National Institutes of Health grants RO1 EB000858 and EB000664.
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