Development of Two Murine Antibodies against Neospora caninum Using Phage Display Technology and Application on the Detection of N. caninum

Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1) and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA) with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

[1]  G. P. Smith,et al.  Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. , 1985, Science.

[2]  C. Milstein,et al.  Derivation of specific antibody‐producing tissue culture and tumor lines by cell fusion , 1976, European journal of immunology.

[3]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[4]  D. Lindsay,et al.  Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. , 1988, Journal of the American Veterinary Medical Association.

[5]  I. Bjerkås,et al.  Unidentified cyst-forming Sporozoon causing encephalomyelitis and myositis in dogs , 2004, Zeitschrift für Parasitenkunde.

[6]  Takahiro Otsuki,et al.  Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins , 2012, BMC Biotechnology.

[7]  L. Sibley,et al.  The p29 and p35 Immunodominant Antigens ofNeospora caninum Tachyzoites Are Homologous to the Family of Surface Antigens of Toxoplasma gondii , 1998, Infection and Immunity.

[8]  M. Reichel,et al.  Progress in the serodiagnosis of Neospora caninum infections of cattle. , 2000, Parasitology today.

[9]  D R Burton,et al.  Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Dubey,et al.  Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay , 1996, Journal of clinical microbiology.

[11]  J. Wastling,et al.  A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera. , 1998, Veterinary parasitology.

[12]  H. Ueda,et al.  Antibody Fab display system that can perform open-sandwich ELISA. , 2009, Analytical biochemistry.

[13]  G. Winter,et al.  Proteolytic selection for protein folding using filamentous bacteriophages. , 1998, Folding & design.

[14]  J. Bye,et al.  Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. , 1996, Journal of molecular biology.

[15]  D. Lindsay,et al.  Fatal congenital Neospora caninum infection in a lamb. , 1990, The Journal of parasitology.

[16]  G. Schares,et al.  Development of an indirect ELISA test using a purified tachyzoite surface antigen SAG1 for sero-diagnosis of canine Toxoplasma gondii infection. , 2009, Veterinary parasitology.

[17]  G. Schares,et al.  Epidemiology and Control of Neosporosis and Neospora caninum , 2007, Clinical Microbiology Reviews.

[18]  D R Burton,et al.  CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. , 1995, Journal of molecular biology.

[19]  A. Guglielmone,et al.  Neospora caninum infections in bovine foetuses and dairy cows with abortions in Argentina. , 1999, International journal for parasitology.

[20]  Janice M. Reichert,et al.  Development trends for monoclonal antibody cancer therapeutics , 2007, Nature Reviews Drug Discovery.

[21]  D. Lindsay,et al.  Abortions, fetal death, and stillbirths in pregnant pygmy goats inoculated with tachyzoites of Neospora caninum. , 1995, American journal of veterinary research.

[22]  A. Hemphill,et al.  Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites. , 2008, International journal for parasitology.

[23]  D. Buxton,et al.  An IgG Avidity ELISA to Discriminate between Recent and Chronic Neospora Caninum Infection , 1999, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[24]  D. Buxton,et al.  Experimental infection of non-pregnant and pregnant sheep with Neospora caninum. , 1997, Journal of comparative pathology.

[25]  P. Conrad,et al.  A Modified Agglutination Test for Neospora caninum: Development, Optimization, and Comparison to the Indirect Fluorescent-Antibody Test and Enzyme-Linked Immunosorbent Assay , 1998, Clinical Diagnostic Laboratory Immunology.

[26]  C. Amemiya,et al.  Phylogenetic diversification of immunoglobulin genes and the antibody repertoire. , 1993, Molecular biology and evolution.