Matrix M protein combined with nucleocapsid N protein could be a promising combination of virus antigens for diagnosing the porcine reproductive and respiratory syndrome. The goal of this work was to express the recombinant M protein of the porcine reproductive and respiratory syndrome virus in Escherichia coli cells and compare its serological reactivity with the N protein of the virus. The gene coding for the M protein was cloned into the pDest17 vector. The resulting protein was purified by metalochelating affinity chromatography. Recombinant M protein was applied as an antigen in immunoblot test and compared on a panel of porcine sera with N protein based IDEXX test. Of 120 examined samples, the majority (78.3%) gave identical results using both compared tests. From the group of discrepant results, IDEXX test identified considerably more positive sera (17.5%) than M protein based test (4.2%). The main contribution of the work is finding that although IDEXX test proved to be more sensitive than M protein based test, 4.2% of sera would escape detection by serological test based on N protein. Further development and purification of the M protein for the use in Enzyme Linked Immunosorbent Assay format test could increase the performance of serological testing.
Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.
- MeSH
- Financing, Government MeSH
- Microscopy, Fluorescence methods utilization MeSH
- Glycoproteins genetics immunology MeSH
- Herpesvirus 1, Equid immunology MeSH
- Immunoblotting methods utilization MeSH
- Antibodies, Viral genetics immunology isolation & purification MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
A phage antibody display library of single chain fragment variables (scFv) was applied to develop anti-equid herpesvirus-1 (EHV-1) glycoprotein D (gD) neutralizing antibodies. To enrich for specific scFvs, the phage antibody library was panned against epitope derived from the N-terminal part of EHV-1 gD. Unique clones were differentiated by BstNI fingerprinting and further characterized by sequencing and immunoreactivity. The neutralizing effect of each clone was assessed by plaque reduction assay. Three clones with neutralizing effect were isolated.
- MeSH
- Cell Line MeSH
- Financing, Organized MeSH
- Microscopy, Fluorescence MeSH
- Herpesvirus 1, Equid immunology MeSH
- Herpesviridae Infections immunology veterinary MeSH
- Immunoblotting MeSH
- Immunoglobulin Fragments immunology MeSH
- Kinetics MeSH
- Horses MeSH
- Molecular Sequence Data MeSH
- Horse Diseases virology MeSH
- Peptide Library MeSH
- Viral Plaque Assay MeSH
- Viral Envelope Proteins immunology MeSH
- Recombinant Proteins immunology MeSH
- Amino Acid Sequence MeSH
- Immunoglobulin Variable Region immunology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
Antimicrobial agent abuse poses a serious threat for future pharmacotherapy, including vaginal administration. The solution can be found in simple polymeric systems with inherent antimicrobial properties without the need to incorporate drugs, for instance alginate beads cross-linked by bivalent ions. The main goal of the presented study was to provide improvement on the well-documented cytotoxicity of Cu2+ cross-linked alginate. Alginate beads were prepared by external ionotropic gelation by cross-linking with Cu2+, Ca2+ and Zn2+ ions, separately and in mixtures. Morphological properties, swelling capacity, ion release and efficacy against the most common vaginal pathogens (C. albicans, E. coli, E. faecalis and virus strain-human herpesvirus type 1) were evaluated. The prepared particles (particle size 1455.68 ± 18.71-1756.31 ± 16.58 µm) had very good sphericity (0.86 ± 0.04-0.97 ± 0.06). In mixture samples, Cu2+ hampered second ion loading, and was also released incompletely (18.75-44.8%) compared to the single ion Cu2+ sample (71.4%). Efficacy against the selected pathogens was confirmed in almost all samples. Although anticipating otherwise, ion mixture samples did not show betterment over a Cu2+ cross-linked sample in cytotoxicity-pathogen efficacy relation. However, the desired improvement was found in a single ion Zn2+ sample whose minimal inhibition concentrations against the pathogens (0.6-6.12 mM) were close to, or in the same mathematical order as, its toxic concentration of 50 (1.891 mM). In summary, these findings combined with alginate's biocompatibility and biodegradability give the combination solid potential in antimicrobial use.
- Publication type
- Journal Article MeSH
