BACKGROUND: Without appropriate culture systems for hepatitis E virus (HEV), sufficient natural viral proteins are difficult to generate for use in serological tests. Therefore, it is important to produce large amounts of HEV recombinant proteins in an economical way. The present study developed ELISAs using 2 truncated forms of the HEV open reading frame (ORF) 2 protein in order to detect anti-HEV IgG in serum samples. METHODS: Two truncated forms of the ORF2 protein were expressed in Escherichia coli and were purified by Ni2+-chelate-affinity chromatography (Qiagen, Germany). Two ELISAs were developed using these proteins and were compared with DIA.PRO HEV IgG ELISA kit (DIA.PRO. Italy) in 220 serum samples. RESULTS: High yields of the target proteins were obtained through codon optimization. The concentration and purity of the proteins were improved with Amicon filters (EMD Millipore, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis of the resultant proteins showed a protein band of approximately 60 kDa corresponding to ORF2.1 (amino acids 112-660) and a protein band of approximately 55 kDa corresponding to ORF2.2 (amino acids 112-607). Positive agreement, negative agreement, and concordance of the 2 in-house ELISAs compared with DIA.PRO HEV IgG ELISA kit were 87%, 99.5%, and 98.1%, respectively (kappa=0.899, P=0.625). CONCLUSIONS: The newly developed ELISAs are useful for detecting anti-HEV IgG in serum samples and are highly concordant with DIA.PRO HEV IgG ELISA kit.
Amino Acid Sequence ; Antibodies/*blood ; *Enzyme-Linked Immunosorbent Assay ; Escherichia coli/metabolism ; Hepatitis E virus/*metabolism ; Humans ; Immunoglobulin G/*blood ; Molecular Sequence Data ; Recombinant Proteins/biosynthesis/immunology/isolation & purification ; Sequence Alignment ; Viral Proteins/chemistry/*immunology/metabolism

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

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.
Adult ; Aged ; Antibodies, Viral/*blood ; Antigens, Viral/genetics/immunology/metabolism ; Capsid Proteins/genetics/immunology/metabolism ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin A/blood ; Immunoglobulin G/blood ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Recombinant Proteins/biosynthesis/genetics/immunology ; Republic of Korea/epidemiology ; Rotavirus/isolation & purification/*metabolism ; Rotavirus Infections/*epidemiology/virology ; Seroepidemiologic Studies ; Time Factors ; Young Adult

OBJECTIVETo express and purify Hap protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic system, and study its immunogenicity and adhesive activity.

METHODHap protein was expressed in E.coli BL21 with pET32a (+)-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit (CT-B), and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay.

RESULTSSDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM (P<0.01). Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice (P<0.05).

CONCLUSIONHighly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.

Adhesiveness ; Animals ; Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; immunology ; Cholera Toxin ; immunology ; Escherichia coli ; genetics ; metabolism ; Haemophilus Infections ; prevention & control ; Haemophilus influenzae ; metabolism ; Immunization ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Serine Endopeptidases ; biosynthesis ; genetics ; immunology

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

VP1 is a major antigenic protein of foot-and-mouth disease virus(FMDV), which induces the immune response against FMDV infection, and contains several epitopes of the virus. We designed and chemically synthesized a DNA fragment which encoding a tandem repeat protein of 136-160aa and 198-211aa of a strain of type Asia I FMDV, and cloned the gene of heavy chain constant region of sheep IgG. By using the BamH I, EcoR I and Xho I sites, both genes were cloned into pPROExHTb vector in turn to form a recombinant plasmid pPRO-FshIgG A chimeric protein, named FshIgG, was obtained after transforming the pPRO-FshIgG into Escherichia coli BL21 (DE3) host cell and induced by IPTG. Inoculation with 100 microg FsIgG induced strong neutralizing antibody response in guinea pigs, and FshIgG inoculated guinea pigs were also protected against 200 ID50 FMDV challenge. Our study indicated that the heavy chain constant region of sheep IgG can act as the carrier protein for FMDV peptide epitopes, and FshIgG is a potential multiepitope peptide vaccine candidate to prevent FMDV infection.
Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; biosynthesis ; genetics ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Genetic Vectors ; genetics ; Guinea Pigs ; Immunization ; Immunoglobulin G ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sheep ; Viral Vaccines ; genetics ; immunology

The mRNA expression of several cytokines was evaluated in splenocytes and mesenteric lymph node (MLN) cells of rats infected with Capillaria hepatica by reverse-transcription (RT)-PCR until week 12 after infection. IgG1 and IgG2a, which are associated with Th1 and Th2 response, respectively, were also assessed by ELISA. The results indicated that the majority of cytokines, including the Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4, IL-5 and IL-10) were expressed at maximal levels during the early stage of infection (after week 1-2), and the ELISA data also evidenced a similar pattern of changes in IgG1 and IgG2a. Th1 and Th2 cytokines responded in a similar fashion in this rat model. The expression of cytokines in splenocytes was significantly higher than that in MLN cells, thereby indicating that cytokine production is controlled more by spleen than by MLN. In addition, the observation that IFN-gamma expression increased unexpectedly at the time of maximal egg production (6 weeks after infection) indicated that IFN-gamma is a cytokine reacting against egg production. However, increased IL-5 expression occurring in tandem with worm activity indicated that the activity of C. hepatica might be controlled by IL-5 expression.
Animals ; Antibodies, Helminth/*blood ; Capillaria/*immunology ; Cytokines/*biosynthesis/genetics ; Disease Models, Animal ; Enoplida Infections/*immunology ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Immunoglobulin G/*blood ; Lymph Nodes/immunology ; Lymphocytes/immunology ; RNA, Messenger/analysis/genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen/cytology/immunology ; Th1 Cells/immunology ; Th2 Cells/immunology

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.
Animals ; Antibodies, Protozoan/*biosynthesis/blood ; Cryptosporidiosis/*immunology ; Cryptosporidium parvum/*immunology/isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Feces/parasitology ; Female ; Immunocompromised Host ; Immunoglobulin G/biosynthesis/blood ; Immunoglobulin M/biosynthesis/blood ; Mice ; Mice, Inbred C57BL ; Oocysts/immunology ; Specific Pathogen-Free Organisms ; Time Factors

OBJECTIVETo investigate the effects of glutamine enriched enteral feeding on immunoregulation in burn patients.

METHODSTwenty burn patients were randomly divided into enteral nutrition (EN) group and enteral immune nutrition (EIN) group, with 12 patients in each group. Patients in EN group received a standard enteral formula, while those in EIN group received an enteral formula enriched with glutamine after hospital admission. Nutritional support was continued for 10 days. Blood samples were obtained to determine plasma level of total protein (TP), albumin (ALB), prealbumin (PAB) and transferrin (TF) at 1, 4, 7, 10 post-burn days (PBD). At the same time the concentration of immunoglobulin (IgA, IgG and IgM) were determined, the percentage of CD3+, CD4+, CD8+ subpopulations of T lymphocytes, and the ratio of CD4+/CD8+ were determined by FCM.

RESULTS(1) There were no obvious difference of the plasma level of TP, ALB, TF, CD3+, IgM between the two groups at each time-point (P > 0.05). (2) The plasma PAB contents in EIN group were significant higher than that in EN group on 4 PBD [(90 +/- 14 vs 60 +/- 15) mg/L, P < 0.05], 7 PBD [(92 +/- 16 vs 64 +/- 13) mg/L, P < 0.05] and 10 PBD [(106 +/- 21 vs 72 +/-16) mg/L, P < 0.05]. (3) The percentage of CD4+ subpopulation and ratio of CD4+/CD8+ in EIN group were obviously higher than those in EN group on 7 PBD [CD4+ (55 +/- 5 vs 45 +/- 5)%, CD4+/CD8+ (1.92 +/- 0.31 vs 1.53 +/- 0.27)%, P < 0.05] and 10 PBD [CD4+ (56 +/- 5 vs 49 +/- 5)%, CD4+/CD8+ (2.36 +/- 0.36 vs 1.72 +/- 0.42), P < 0.05]. (4) The concentration of IgA and IgG in EIN group were markedly higher than that in EN group on 7 PBD [IgA (2.8 +/- 0.6 vs 2.2 +/- 0.5) g/L, IgG (12.1 +/- 1.3 vs 9.8 +/- 1.2) g/L, P < 0.05] and 10 PBD [IgA (3.1 +/- 0.6 vs 2.5 +/- 0.5) g/L, IgG (14.2 +/- 1.3 vs 10.4 +/- 1.3) g/L, P < 0.05].

CONCLUSIONThese findings suggest that glutamine enriched enteral feeding can improve nutritional status by promoting the synthesis of IgA, IgG, and increasing the PAB concentration, and corrected immunologic dysfunction in burn patients.

Adolescent ; Adult ; Burns ; blood ; immunology ; therapy ; Enteral Nutrition ; Female ; Glutamine ; therapeutic use ; Humans ; Immunoglobulin A ; biosynthesis ; Immunoglobulin G ; biosynthesis ; Male ; Middle Aged ; Prealbumin ; metabolism ; T-Lymphocyte Subsets ; immunology ; Young Adult

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.
Protozoan Proteins/immunology/*isolation & purification ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Life Cycle Stages/immunology ; Leishmaniasis, Cutaneous/immunology ; Leishmania major/*immunology ; Immunoglobulin G/*biosynthesis/blood ; Humans ; Female ; Blotting, Western/methods ; Antigens, Protozoan/immunology/*isolation & purification ; Animals