The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.
Animals ; Antibodies, Monoclonal/*biosynthesis/immunology ; Chromatography, Affinity/veterinary ; Electrophoresis, Gel, Two-Dimensional/veterinary ; Immunoblotting/veterinary ; Immunoglobulin Heavy Chains/immunology ; Immunoglobulin Light Chains/immunology ; Immunoglobulins/blood/*immunology ; Perciformes/blood/*immunology

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 microg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.
Animals ; Antigens/immunology/isolation & purification ; Argasidae/immunology ; Chromatography, Affinity/veterinary ; Electrophoresis, Polyacrylamide Gel/veterinary ; Female ; Glycoproteins/*immunology/isolation & purification ; Immunoblotting/veterinary ; Injections, Intramuscular/veterinary ; Ixodidae/growth & development/*immunology ; Life Cycle Stages ; Male ; Rabbits/*immunology/parasitology ; Reproduction ; Species Specificity ; Tick Infestations/immunology/prevention & control/*veterinary

The present study was conducted to identify new target immunogenic molecules from the larval stage of the cattle tick, Boophilus annulatus (Acari: Ixodidae). Two specific larval glycoproteins (GLPs) were isolated by two-step affinity chromatography. The larval immunogens were first purified with CNBr-Sepharose coupled to rabbit anti-larval immunoglobulins, and the glycoproteins were then purified with Con-A Sepharose. These glycoproteins have molecular weights of approximately 32 and 15 kDa with isoelectric points between 6.8 and 7.2. Antibodies against the two GLPs, labeled I and II, were detected in the anti-whole tick, -whole larval, and -gut antigens through immunoblot analysis. These results suggest that these GLPs are good immunogens and can be useful in the vaccination of cattle against tick infestation.
Amino Acid Sequence ; Animals ; Cattle ; Cattle Diseases/immunology/*parasitology/prevention & control ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Glycoproteins/immunology/*isolation & purification ; Immunoblotting ; Isoelectric Focusing ; Ixodidae/chemistry/*immunology ; Male ; Molecular Weight ; Rabbits ; Sequence Analysis, Protein ; Tick Infestations/immunology/parasitology/prevention & control/*veterinary

Rapid serodiagnostic methods for Toxoplasma gondii infection in cats are urgently needed for effective control of transmission routes toward human infections. In this work, 4 recombinant T. gondii antigens (SAG1, SAG2, GRA3, and GRA6) were produced and tested for the development of rapid diagnostic test (RDT). The proteins were expressed in Escherichia coli, affinity-purified, and applied onto the nitrocellulose membrane of the test strip. The recombinant SAG1 (rSAG1) showed the strongest antigenic activity and highest specificity among them. We also performed clinical evaluation of the rSAG1-loaded RDT in 182 cat sera (55 household and 127 stray cats). The kit showed 0.88 of kappa value comparing with a commercialized ELISA kit, which indicated a significant correlation between rSAG1-loaded RDT and the ELISA kit. The overall sensitivity and specificity of the RDT were 100% (23/23) and 99.4% (158/159), respectively. The rSAG1-loaded RDT is rapid, easy to use, and highly accurate. Thus, it would be a suitable diagnostic tool for rapid detection of antibodies in T. gondii-infected cats under field conditions.
Animals ; Antigens, Protozoan/*diagnostic use/genetics ; Cat Diseases/*diagnosis ; Cats ; Chromatography, Affinity ; Escherichia coli/genetics ; *Point-of-Care Systems ; Protozoan Proteins/*diagnostic use/genetics ; Recombinant Proteins/diagnostic use/genetics ; Sensitivity and Specificity ; Serologic Tests/methods ; Toxoplasma/genetics ; Toxoplasmosis, Animal/*diagnosis ; Veterinary Medicine/*methods