Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.
Animals ; Antibodies, Viral/*blood ; Antigens, Viral/*diagnostic use/genetics/metabolism ; Baculoviridae/genetics ; Chickens ; HN Protein/*diagnostic use/genetics/metabolism ; Hemagglutination Inhibition Tests/*methods/veterinary ; Newcastle Disease/*diagnosis/immunology/virology ; Newcastle disease virus/genetics/*immunology/metabolism ; Poultry Diseases/*diagnosis/immunology/virology ; Recombinant Proteins/diagnostic use/genetics/metabolism ; Sf9 Cells ; Spodoptera

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.
Antibodies, Bacterial/blood/chemistry/immunology ; Antigens, Bacterial/diagnostic use/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gold/chemistry ; Humans ; Immunoassay ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Orientia tsutsugamushi/immunology/*metabolism ; Recombinant Proteins/biosynthesis/diagnostic use/genetics ; Scrub Typhus/*diagnosis ; Sensitivity and Specificity ; Serotyping

Interleukin-2 plays an important role in T lymphocyte proliferation and immune response regulations. In this study, porcine IL-2 cDNA was cloned from peripheral blood mononuclear cells, and recombinant porcine IL-2 (rpIL-2) was expressed in Escherichia coli. The size of rpIL-2 without signal peptides was about 15 kDa when determined by SDS-PAGE and Western blotting analysis. Anti-rpIL-2 antibody was produced from mice immunized with the purified rpIL-2, and its specificity was examined by Western blotting and ELISA. In the Western blotting assay, anti-rpIL-2 and anti-recombinant human IL-2 (rhIL-2) antibodies specifically recognized rpIL-2 and rhIL-2, respectively. However, anti-rpIL-2 antibody did not recognize rhIL-2, and anti-rhIL-2 antibody also did not react with rpIL-2 in the same assay. In ELISA, anti-rpIL-2 antibody strongly interacted with both rpIL-2 and rhIL-2, and anti-rhIL-2 antibody also efficiently recognized both proteins. Taken together, the specificity of anti-rpIL-2 antibody for rpIL-2 was demonstrated by Western blotting and ELISA. It was also shown that ELISA is more efficient than Western blotting in determining the species cross-reactivity of anti-rpIL-2 antibody.
Animals ; Antibodies/diagnostic use/*metabolism ; Antibody Specificity ; Blotting, Western/veterinary ; Cloning, Molecular ; Cross Reactions ; Electrophoresis, Polyacrylamide Gel/veterinary ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Escherichia coli/genetics/metabolism ; Humans ; Interleukin-2/biosynthesis/genetics/*immunology ; Mice ; Recombinant Proteins/biosynthesis/genetics/immunology ; Species Specificity ; Swine