Nanocrystal Quantum Dot-Conjugated Anti-Myeloperoxidase Antibody as the Detector of Activated Neutrophils

Abstract

Fluorescent nanocrystal quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. Here we show that QDs conjugated with antibody against neutrophil peroxidase, myeloperoxidase (MPO). We designed a novel method to conjugate QDs to antibody without losing any antibody function including their antigen recognizing and Fc-receptor binding activities. When we applied anti-MPO antibody (Ab) with conventional organic probes in the case of immunostaining of living cells, the antibodies lost their fluorescence because of MPO enzymic activity to produce reactive oxygen species. Our QD-conjugated anti-MPO (alpha-MPO-QDs) can detect MPO on the surface of activated neutrophils. In addition, anti-MPO-QDs did not react to the inactivated neutrophils. In conclusion, we demonstrated that antibody visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. Taken together, these techniques have the possibility that QDs can reveal the activation of neutrophils by immunostaining and flow cytometric analysis as a powerful tool for diagnosis of the neutrophil activation in vitro.

Trafficking of QD‐Conjugated MPO‐ANCA in Murine Systemic Vasculitis and Glomerulonephritis Model Mice

“…5b). In addition, MPO translocation on neutrophils was observed at a concentration of 10 Ϫ8 M FMLP, that is consistent with the result that human neutrophils show chemoattraction on FMLP at 10 Ϫ8 M (18). However, CAWS stimulation induces no MPO translocation (Fig.…”

“…The antibody captured on QD measured with the Bradford reagent (BioRad Laboratories, Hercules, Calif., U.S.A.) with the standard curve of bovine serum albumin indicated that each ANCA-QD had approximately 8.5 Abs per QD nanocrystal (data not shown, see ref. 18). The function of ANCA-QD was checked by fluorescent Western blotting and histochemical analysis (39).…”

MPO-ANCA induces IL-17 production by activated neutrophils in vitro via its Fc region- and complement-dependent manner

“…These observations are consistent with our results that neutrophils also produce IL-23 and mediate the Th 17 -medicated immune response. The higher ANCA titer is probably established by repeated MPO leakage on the membrane surface due to repeated or chronic fungal infection [28]. Th 17 and IL-17-mediated immune responses are required for the appropriate immunity for the prevention of fungal infection, but unfortunately in some cases it facilitates the resting Th 17 -mediated immune response while activating the neutrophils inappropriately via excessive proinflammatory cytokine induction, thus finally resulting in an autoimmune disorder.…”

“…4a). To test whether this separation process activates the neutrophils, we confirmed the surface expression of MPOs, whose expression is observed only on the membrane surface of activated neutrophils [28]. The surface expression of MPO is not observed in purified peritoneal neutrophils (Fig.…”

Evaluation of Anti-Inflammatory Drug-Conjugated Silicon Quantum Dots: Their Cytotoxicity and Biological Effect

“…Cadmium-based QDs are of special interest because they have unique photonic properties and photonic stability [6], and they are currently being used in fluorescent biodetection systems [7–9] including bio-imaging performed in vitro [10,11] and in vivo [12–15]. Our group investigated the efficacy of the QDs modified with various chemicals, antibodies and ligands as specific biological probes [16–19]. …”