Antibodies against citrinin (CTN) were generated from rabbits, which were injected with CTN-keyhole limpet hemocyanin (KLH). This work involved the development of a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and a rapid gold nanoparticle immunochromatographic strip (immunostrip) method for analyzing CTN in Monascus-fermented food. CTN at a concentration of 5.0 ng/mL caused 50% inhibition (IC50) of CTN-horseradish peroxidase (CTN-HRP) binding to the antibodies in the cdELISA. The capable on-site detection of CTN was accomplished by a rapid antibody-gold nanoparticle immunostrip with a detection limit of 20 ng/mL and that was completed within 15 min. A close inspection of 19 Monascus-fermented foods by cdELISA confirmed that 14 were contaminated with citrinin at levels from 28.6–9454 ng/g. Further analysis with the immunostrip is consistent with those results obtained using cdELISA. Both means are sensitive enough for the rapid examination of CTN in Monascus-fermented food products.

No abstract

Due to its highly immunogenic nature and the great engineerability, filamentous phage has shown promising antitumor activities in preclinical studies. Previous designs of antitumor phage mainly focused on tumor targeting using a cancerspecific moiety displayed on the minor capsid protein, pIII. In this work, we developed a new therapeutic platform of filamentous phage, in which the major capsid protein pVIII was utilized for displaying an antitumor cytokine. We showcased that a 16.1-kD cytokine GM-CSF could be efficiently presented on the M13 phage particle using the 8 + 8 type display system through a highly tolerable pVIII variant P8(1a). We verified that the GM-CSF phage was a potent activator for STAT5 signaling in murine macrophage. The GM-CSF phage significantly reduced the tumor size by more than 50% as compared to the unmodified phage in a murine colorectal cancer model. Immunological profiling of the tumor-infiltrating leukocytes revealed that an increase of CD4+ lymphocytes in the GM-CSF phage treatment group. Furthermore, the combined therapy of the GM-CSF phage and radiation greatly improved the therapeutic potency with a 100% survival rate and a 25% complete remission rate. We observed that the IFN-γ expression was dramatically up-regulated by the combined therapy in multiple types of tumor-infiltrating immune cells. Overall, we created a novel vehicle for cytokine therapy using the pVIII filamentous phage display. This new platform can be multiplexed with other phage engineering approaches, such as displaying targeting ligands on pIII or encapsulating therapeutic genes inside phage capsids, to create multifunctional nanoparticles for cancer therapy.