Low-Fouling Magnetic Nanoparticles and Evaluation of Their Potential Application as Disease Markers Assay in Whole Serum

A novel chondroitin sulfate (CSA) coated magnetic nanoparticle, with almost perfect antifouling properties in a variety of external environments, especially in complex biological systems, was successfully prepared. This is the first report on the utilization of CSA for the construction of an antifouling surface of magnetic nanoparticles. More encouragingly, probes based on these nanoparticles have successfully shown great applied potency in the rapid and direct diagnosis of disease markers in the whole blood

Morphometric analysis of muscles.

<p>(A) H&E staining displayed myofiber hypertrophy in muscles of M17 compared with control sheep. (B) Distribution of muscle fiber sizes in M17 and control sheep. A total of 500 fibers from each sheep were measured. (C) Mean myofiber diameter for M17 and controls.</p

Summary of SCNT results.

<p>Summary of SCNT results.</p

Transgene integration and expression analysis in three cloned lambs.

<p>PCR analysis using primers specific for the shRNA expression cassette (A) and Neo (B). (C) Expression of the shRNA targeting MSTN in muscle tissues of transgenic sheep M17, M18 and M23. Negative control (NC): control sheep; positive control (PC): ploxP-shMSTN3 vector. M: Maker; TF-s2: Transgenic cell clone TF-s2; TF-s19: Transgenic cell clone TF-s19. (D) Western blot analysis of MSTN protein expression in muscle tissues of transgenic sheep M17, M18 and M23 and control sheep (Ctr1, Ctr2 and Ctr3).</p

Body weight of transgenic sheep and controls.

<p>M17, M18, M23 and three control sheep were weighted at 1, 20, 40, 60 and 90 days after birth. Control values are average weights of three control sheep.</p

Schematic illustration representing ploxP-shMSTN3 vector used in this study.

<p>Loxp: recombination site of Cre recombinase for bacteriophage P1; CMV: CMV promoter; Neo: neomycin gene; U6: polymerase III U6-RNA gene promoter, shRNA: short hairpin RNA. Arrowhead indicated localization of the primers specific for shRNA expression cassette and Neo gene. The size of the PCR amplicons is indicated.</p

Expression levels of MHCII, MyoD, Myogenin and Smad2 in the muscle tissues of transgenic sheep.

<p>mRNA expression of MHCII (A), MyoD (B), Myogenin (C) and Smad2 (D) were determined using Real-time RT-PCR and normalized to GAPDH expression. * P<0.05, ** P<0.01.</p

Multiple amino acid sequence alignment of BVDV-E2 nanobody clones.

<p>The framework and CDR regions and amino acid numbering were performed as stipulated in Gene.DOC. The CDR regions outlined in lines. The CDR regions are outlined in lines. Sequencing analysis indicated that the nanobody clones were highly homologous to the camel VHH sequence.</p

Monoclonal phage ELISA.

<p>A total of nine clones (out of 96) were analyzed with monoclonal phage ELISA. BVDV-E2 antigens at 10 μg/ml were coated in each well. PBS served as the negative control. A total of five clones were selected on the basis of absorbance. The x-axis presents the clone number, and the y-axis shows the absorbance values at 450 nm.</p

Monoclonal phage ELISA.

<p>A total of 96 random clones from the library were analyzed with monoclonal phage ELISA. BVDV-E2 antigens at 10 μg/ml were coated in each well. PBS served as the negative control and M13K07 was used as the positive control. A total of nine clones were selected on the basis of absorbance. The x-axis shows the number of clones, and the y-axis shows the absorbance values at 450 nm.</p