Anti-PcrV mAbs (University of Cologne)

The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extraction of viral pathogens from a sample in a chip-scale, sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV mAbs. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. The ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter molecules and then a layer of mAbs. The Raman reporter molecule is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to selectively tag substrate-bound FCV. This paper describes the development of the assay, which uses cell culture media as a sample matrix and has a linear dynamic range of 1 x 10(6)-2.5 x 10(8) viruses/mL and a limit of detection of 1 x 10(6) viruses/mL. These results reflect the findings from a detailed series of investigations on the effects of several experimental parameters (e.g., salt concentration, ERL binding buffer, and sample agitation), all of which were aimed at minimizing nonspecific binding and maximizing FCV binding efficiency. The performance of the assay is correlated with the number of captured FCV, determined by atomic force microscopy, as a means of method validation.

Acute lung injury caused by Pseudomonas aeruginosa is attributed to the translocation of cytotoxin into pulmonary epithelial cells via the P. aeruginosa type III secretion system. This virulence can be blocked with a specific antibody against PcrV in this secretion system. However, because anti-PcrV antibodies do not have bactericidal activity, the treatment of bacteria depends on the phagocytic system of the host. In this study, we investigated the therapeutic effect of combination therapy with an anti-PcrV antibody and bactericidal bacteriophages on acute lung injury and subsequent death in mice compared with a single treatment. After the mice intratracheally received a lethal dose of the cytotoxic P. aeruginosa strain, a second instillation was performed with saline, anti-PcrV IgG, bacteriophages, or a mixture of anti-PcrV and bacteriophages. The survival rates 24 h after infection were as follows: 7.1% in the saline group, 26.7% in the anti-PcrV group, 41.2% in the phage group, and 66.7% in the anti-PcrV + phage group ( P < 0.001 vs saline-treated group). The activity of surviving mice in the anti-PcrV + phage group was significantly greater than that in the saline group. The lung weight in the anti-PcrV + phage group was significantly lower than that in the anti-PcrV group. In conclusion, combination therapy with an anti-PcrV antibody and a bacteriophage reduces acute lung injury and suggests improved survival compared with each treatment alone. This combination therapy, which does not rely on conventional antibiotics, could constitute a new strategy for treating multidrug-resistant P. aeruginosa infections.

Combination therapy with either bacteriophages alone or in combination with anti-PcrV antibodies in a mouse model of Pseudomonas aeruginosa pneumonia may reduce the acute lung injury and improve survival. This combination therapy, which does not rely on conventional antibiotics, may be a new strategy to treat multidrug-resistant Pseudomonas aeruginosa infections.