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A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting.

Wang SM, He X, Li N, Yu F, Hu Y, Wang LS, Zhang P, Du YK, Du SS, Yin ZF, Wei YR, Mulet X, Coia G, Weng D, He JH, Wu M, Li HP - Int J Nanomedicine (2015)

Bottom Line: Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17.Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity.Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People's Republic of China.

ABSTRACT
Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies' potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

No MeSH data available.


Related in: MedlinePlus

Detection of rSPA by Western immunoblot and ELISA.Notes: (A) Western immunoblot showed the expressed rSPA (lane 2) can bind to a goat anti-rSPA polyclonal antibody compared to the negative control group (lane 1) at 27 kDa. (B) ELISA analysis showed a high binding activity of the expressed rSPA (lane 2) to anti-rSPA polyclonal antibody compared to the negative control (lane 1). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.
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f1-ijn-10-2857: Detection of rSPA by Western immunoblot and ELISA.Notes: (A) Western immunoblot showed the expressed rSPA (lane 2) can bind to a goat anti-rSPA polyclonal antibody compared to the negative control group (lane 1) at 27 kDa. (B) ELISA analysis showed a high binding activity of the expressed rSPA (lane 2) to anti-rSPA polyclonal antibody compared to the negative control (lane 1). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.

Mentions: The recombinant vector pET-44a-rSPA containing a 245-amino acid fragment of SPA was created as described in the Materials and methods section and transformed into BL21 E. coli. The optimum expression condition of the recombinant protein was found to be 0.8 mM IPTG at 37°C for 6 hours. The molecular weight of the expressed protein was estimated to be approximately 27 kDa, suggesting a correct size of typical rSPA protein. Immunoblotting analysis demonstrated that the expressed protein specifically bound to anti-rSPA polyclonal antibody at 27 kDa, whereas no band was seen in a control GST protein (Figure 1A). ELISA also showed robust binding activity between rSPA and goat anti-rSPA-poly-ant compared to the control protein (Figure 1B). Overall data suggest that we successfully constructed, produced, and purified rSPA.


A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting.

Wang SM, He X, Li N, Yu F, Hu Y, Wang LS, Zhang P, Du YK, Du SS, Yin ZF, Wei YR, Mulet X, Coia G, Weng D, He JH, Wu M, Li HP - Int J Nanomedicine (2015)

Detection of rSPA by Western immunoblot and ELISA.Notes: (A) Western immunoblot showed the expressed rSPA (lane 2) can bind to a goat anti-rSPA polyclonal antibody compared to the negative control group (lane 1) at 27 kDa. (B) ELISA analysis showed a high binding activity of the expressed rSPA (lane 2) to anti-rSPA polyclonal antibody compared to the negative control (lane 1). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403696&req=5

f1-ijn-10-2857: Detection of rSPA by Western immunoblot and ELISA.Notes: (A) Western immunoblot showed the expressed rSPA (lane 2) can bind to a goat anti-rSPA polyclonal antibody compared to the negative control group (lane 1) at 27 kDa. (B) ELISA analysis showed a high binding activity of the expressed rSPA (lane 2) to anti-rSPA polyclonal antibody compared to the negative control (lane 1). *P<0.05.Abbreviations: rSPA, rat surfactant protein A; ELISA, enzyme-linked immunosorbent assay; OD, optical density.
Mentions: The recombinant vector pET-44a-rSPA containing a 245-amino acid fragment of SPA was created as described in the Materials and methods section and transformed into BL21 E. coli. The optimum expression condition of the recombinant protein was found to be 0.8 mM IPTG at 37°C for 6 hours. The molecular weight of the expressed protein was estimated to be approximately 27 kDa, suggesting a correct size of typical rSPA protein. Immunoblotting analysis demonstrated that the expressed protein specifically bound to anti-rSPA polyclonal antibody at 27 kDa, whereas no band was seen in a control GST protein (Figure 1A). ELISA also showed robust binding activity between rSPA and goat anti-rSPA-poly-ant compared to the control protein (Figure 1B). Overall data suggest that we successfully constructed, produced, and purified rSPA.

Bottom Line: Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17.Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity.Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People's Republic of China.

ABSTRACT
Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies' potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

No MeSH data available.


Related in: MedlinePlus