Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.
Animals ; Antibodies, Anti-Idiotypic/*analysis/blood/genetics ; Capsid Proteins/*genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Hepatitis E/diagnosis/immunology/*veterinary/virology ; Hepatitis E virus/genetics/*isolation & purification/metabolism ; Immunoglobulin G/blood/genetics ; ROC Curve ; Recombinant Proteins/genetics/metabolism ; Swine ; Swine Diseases/*diagnosis/immunology/virology

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA HisBind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.
Animals ; Antibodies, Viral ; analysis ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Cattle ; Cattle Diseases ; diagnosis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; immunology ; Foot-and-Mouth Disease Virus ; chemistry ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-alpha (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses.
Administration, Intranasal ; Animals ; Anisakiasis/*immunology/parasitology ; Anisakis/*immunology/metabolism ; Bronchoalveolar Lavage Fluid ; Chemokines/metabolism ; Cytokines/analysis/*metabolism ; Eosinophils/metabolism ; Female ; Gene Expression Regulation/*immunology ; Helminth Proteins/*immunology ; Hypersensitivity/*immunology/parasitology ; Immunoglobulin E/immunology ; Immunoglobulin G/immunology ; Larva/immunology/metabolism ; Lung/metabolism ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins/immunology ; Th17 Cells/metabolism ; Th2 Cells/metabolism

BACKGROUND: Insulin assays are affected by varying degrees of interference from anti-insulin antibodies (IAs) and by cross-reactivity with recombinant insulin analogues. We evaluated the usefulness of the E170 insulin assay by assessing IA effects and cross-reactivity with 2 analogues. METHODS: Sera were obtained from 59 type 2 diabetes patients receiving continuous subcutaneous insulin infusion and 18 healthy controls. Insulin levels were determined using an E170 analyzer. To investigate the effects of IAs, we performed IA radioimmunoassays, and analyzed the differences between directly measured insulin (direct insulin) and polyethylene glycol (PEG)-treated insulins (free, IA-unbound; total, IA-bound and unbound insulin). We performed in-vitro cross-reactivity tests with insulin aspart and insulin glulisine. RESULTS: In IA-positive patients, E170 free insulin levels measured using the E170 analyzer were significantly lower than the direct insulin levels. The mean value of the direct/free insulin ratio and IA-bound insulin, which were calculated as the difference between total and free insulin, increased significantly as endogenous IA levels increased. The E170 insulin assay showed low cross-reactivities with both analogues (< 0.7%). CONCLUSIONS: IAs interfered with E170 insulin assay, and the extent of interference correlated with the IA levels, which may be attributable to the increase in IA-bound insulin, and not to an error in the assay. The E170 insulin assay may measure only endogenous insulin since cross-reactivity is low. Our results suggest that the measurement of free insulin after PEG pre-treatment could be useful for beta cell function assessment in diabetic patients undergoing insulin therapy.
Adult ; Aged ; Aged, 80 and over ; Cross Reactions ; Diabetes Mellitus, Type 2/blood/immunology ; Female ; Humans ; Infusions, Subcutaneous ; Insulin/analogs & derivatives/*blood/chemistry/immunology ; Insulin Antibodies/*blood ; Male ; Middle Aged ; Polyethylene Glycols/chemistry ; Radioimmunoassay/instrumentation/*methods ; Recombinant Proteins/analysis/immunology/metabolism

Proteins of the complement system are known to interact with many charged substances. We recently characterized binding of C1q and factor H to immobilized and liposomal anionic phospholipids. Factor H inhibited C1q binding to anionic phospholipids, suggesting a role for factor H in regulating activation of the complement classical pathway by anionic phospholipids. To extend this finding, we examined interactions of C1q and factor H with lipid A, a well-characterized activator of the classical pathway. We report that C1q and factor H both bind to immobilized lipid A, lipid A liposomes and intact Escherichia coli TG1. Factor H competes with C1q for binding to these targets. Furthermore, increasing the factor H: C1q molar ratio in serum diminished C4b fixation, indicating that factor H diminishes classical pathway activation. The recombinant forms of the Cterminal, globular heads of C1q A, B and C chains bound to lipid A and E. coli in a manner qualitatively similar to native C1q, confirming that C1q interacts with these targets via its globular head region. These observations reinforce our proposal that factor H has an additional complement regulatory role of down-regulating classical pathway activation in response to certain targets. This is distinct from its role as an alternative pathway down-regulator. We suggest that under physiological conditions, factor H may serve as a downregulator of bacterially-driven inflammatory responses, thereby fine-tuning and balancing the inflammatory response in infections with Gram-negative bacteria.
Binding, Competitive ; immunology ; Complement Activation ; immunology ; Complement C1q ; chemistry ; immunology ; metabolism ; Complement C4b ; analysis ; Complement Factor H ; chemistry ; immunology ; metabolism ; Complement Pathway, Classical ; immunology ; Escherichia coli ; immunology ; metabolism ; Humans ; Iodine Radioisotopes ; Isotope Labeling ; Lipid A ; immunology ; metabolism ; Liposomes ; immunology ; metabolism ; Protein Binding ; immunology ; Recombinant Proteins ; chemistry ; immunology ; metabolism ; Substrate Specificity

OBJECTIVESTo construct and to express a human-mouse chimeric antibody against Aβ peptide involved in Alzheimer disease by genetic antibody engineering with reducing of its human anti-mouse antibody response.

METHODSTotal RNA was extracted from a murine hybridoma cell line that secreted anti-Aβ monoclonal antibody. The entire gene coding heavy and light chains were amplified using RT-PCR and analyzed by Genebank Blast. The chimeric antibody gene was acquired by variable region gene of the monoclonal antibody with constant region gene of human IgG, in which point mutations were incluced by recombinant PCR technology, respectively. The eukaryotic expression vectors established by cloning chimeric antibody genes of the heavy and light chains into 3.1 were co-transfected into COS-7 cells. The expressed products were analyzed using ELISA and immunohistochemistry subsequently.

RESULTSGenebank Blast analysis showed that the entire cloned antibody genes were in accordance with the murine antibody genes. DNA sequencing confirmed that the expression vectors of chimeric antibody were constructed successfully after splicing the variable region and constant region sequences. By co-transfecting COS-7 cells, a chimeric antibody was produced and collected in the culture medium. The antibody was humanized and bound Aβ specifically by ELISA and immunohistochemistry evaluations.

CONCLUSIONSExpression vector of chimeric antibody against Aβ was constructed successfully and expressed in the eukaryotic cells. It provides a solid base for developing diagnostic and therapeutic methods for Alzheimer's disease in clinic and paves a way for a further humanization in the future.

Alzheimer Disease ; immunology ; metabolism ; Amyloid beta-Peptides ; immunology ; metabolism ; Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; COS Cells ; Cell Line ; Cercopithecus aethiops ; Genetic Vectors ; Humans ; Hybridomas ; cytology ; immunology ; Immunoglobulin Heavy Chains ; genetics ; metabolism ; Immunoglobulin Light Chains ; genetics ; metabolism ; Mice ; Point Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, DNA ; Transfection

In this study, we cloned the NS3 gene from bovine viral diarrhea virus (BVDV) VEDEVAC strain. The result showed that the average P-distance of Pestivirus NS3 amino acid sequence was 0.07 and the VEDEVAC strain was classified to BVDV type 1. Using pET-30a(+) as vector and Escherichia coli Rosetta (DE3) as host, we obtained purified recombinant NS3 protein by Ni-NTA affinity chromatography. Western blotting analysis demonstrated that both BVDV positive serum and classical swine fever virus (CSFV) positive serum were able to recognize the recombinant NS3 protein. Indirect-ELISA assay indicated that the protein could be used as detection antigen.
Animals ; Cattle ; Cloning, Molecular ; Diarrhea Viruses, Bovine Viral ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Peptide Hydrolases ; genetics ; immunology ; Phylogeny ; RNA Helicases ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, Protein ; Viral Nonstructural Proteins ; genetics ; immunology

Yeast Elongation protein 3 (yElp3), the catalytic subunit of the multi-subunit histone acetyltransferase elongator complex, is involved in histone acetylation and transcription, exocytosis and tRNA modification. To study the complex function of yElp3 in yeast, we amplified the yElp3 gene fragment encoding 73aa in the N-terminal from plasmid pYES2-yElp3, and then cloned it into pMXB10 to construct the recombinant plasmid pMXB10-yElp3-219. We expressed the fusion protein in E. coli BL21 (DE3), then purified it by chin affinity column, and finally obtained the soluble purified protein (8.0 kD), which was used to immune the rabbits for acquiring antiserum. ELISA and Western blotting indicated that the polyclonal antibody was of high titration and specificity. Chromatin immunoprecipitation (ChIP) assay with this antibody suggested that yhElp3 exerted the transcriptional regulatory function directly through its presence on the SSA3 gene; this might be the reason that it can rescue the delay activation of SSA3 in elp3delta cells.
Amino Acid Sequence ; Antibodies ; analysis ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Fungal ; Histone Acetyltransferases ; biosynthesis ; genetics ; immunology ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; immunology

In order to study the functions of the HSPs (Heat shock proteins) of Eimeria tenella, we cloned a novel gene (which designated EtHSP) coding HSP of Eimeria tenella by RT-PCR and RACE (Rapid-amplification of cDNA ends). The full-length cDNA sequence of EtHSP was 1802 bp, containing a 1455 bp ORF (Open reading frame) (GenBank Accession No. FJ911605) encoding a deduced protein of 484 amino acids. Real-time PCR revealed that the mRNA level of EtHSP was much higher in sporozoites of E. tenella than other developmental stages (unsporulated oocysts, sporulated oocysts and merozoites). We constructed the recombinant plasmids pET28a(+)-EtHSP, then transformed it into E. coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed in included bodies, with peak expression 6 h after induction by IPTG Western blotting revealed that the protein was specifically recognized by polyclonal antibodies against E. tenella, showing that the fusion protein was native antigen.
Animals ; Chickens ; Eimeria tenella ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Heat-Shock Proteins ; genetics ; immunology ; metabolism ; Inclusion Bodies ; metabolism ; Male ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Sequence Analysis, Protein

OBJECTIVETo evaluate whether the D-3-phosphoglycerate dehydrogenase (Phgdh) correlative antibodies is crucial for AIH, we cloned Phgdh cDNA and constructed plasmid, then purified and identified the immunoreactivity of the recombinant protein, and established the enzyme linked immunosorbent assay (ELISA) to detect Phgdh autoantigen correlative antibodies in diagnosis of autoimmune hepatitis.

METHODSThe constructed plasmid was transformed into E. coli. BL21(D3). This fusion protein was purified by Ni-NTA chromatography and its immunoreactivity was identified by SDS-PAGE and Western blot. The ELISA with the fusion protein was established first, then, the Phgdh autoantigen correlative antibodies in serum of patients with AIH (65) and patients with PBC (122) as well as chronic hepatitis B (CHB) (56), chronic hepatitis C (CHC) (117), and normal controls (60) were detected.

RESULTSThe sequence of Phgdh autoantigen gene was the same as the sequence reported on the genebank. The fusion protein was found about 60kD strip on SDS-PAGE. Western blot analysis showed that the fusion protein had immunoreactivity. When analyzing the serum by ELISA, the immune reactivity to Phgdh was detected in 66.15% of patients with AIH, 21.42% of patients with PBC, 12.50% of patients with CHB, 6.83% of patients with CHC, and 3.30% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P less than 0.01).

CONCLUSIONThe Phgdh cDNA is successfully cloned into E. coli BL21 (D3). The frequency of antibodies to Phgdh is much higher in patients with AIH than in patients with PBC, CHB, CHC and normal control. The antibodies to Phgdh may have utility in improved diagnosis of AIH.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Autoantibodies ; analysis ; genetics ; Autoantigens ; immunology ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Genetic Vectors ; Hepatitis B, Chronic ; blood ; diagnosis ; Hepatitis, Autoimmune ; blood ; diagnosis ; Humans ; Liver Cirrhosis, Biliary ; blood ; diagnosis ; Male ; Middle Aged ; Phosphoglycerate Dehydrogenase ; genetics ; immunology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Young Adult