Characterization of immune response of young pigs to porcine circovirus type 2 infection

A longitudinal study was conducted to characterize the immune response of young swine to infection with porcine circovirus type 2 (PCV-2). Five 8-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally and intramuscularly with a field isolate of PCV-2 at a concentration of 10(4) TCID50/mL. Along with monitoring for clinical signs and viremia, serum samples were collected from all pigs at day 0 and thereafter every 7 days postinoculation (PI) until the termination of the study on day 35 PI. No clinical signs were observed in any of the animals during the study period. In all pigs, PCV-2 was detected by polymerase chain reaction (PCR) in serum samples collected on days 7, 14, and 21 PI. Viral DNA and antigens were detected by in situ hybridization and immunohistochemistry in tonsil, spleen, medial iliac lymph nodes, and ileum collected from each pig at the end of the study. Collectively, na?ve young swine were shown to be susceptible to PCV-2. Virus-specific antibody was detected by an indirect fluorescent antibody (IFA) assay on day 14 PI, but virus-neutralizing antibody was not detected until day 28 PI. As neutralizing antibodies developed, cross-reactivity with PCV type 1 (PCV-1) also developed on the IFA test. Western immunoblot analysis revealed three PCV-2 proteins with molecular masses of 28 kd, 28.5 kd, and 35 kd. The 35-kd protein was also demonstrated in PCV-1, suggesting that this protein induced the cross-reactivity between PCV types 1 and 2. Antibody to the 28-kd protein was detected on day 14 PI and later, indicating that this protein was the most immunogenic. Because of its immunogenicity and specificity to PCV-2, and 28-kd protein might provide the antigenic basis for the development of diagnostic tests for detection of PCV-2 antibody.     

Development and application of a double-antigen sandwich enzyme-linked immunosorbent assay for detection of antibodies to porcine circovirus 2

A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.     

Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization

Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. Rabbit sera selected from positive AGID test data were used to standardize the method. Briefly, wells of flexible microtitre plates coated with antigen were incubated with antiserum followed by a secondary labelled antibody. The radioactivity remaining in the wells after washing provided a measure of the amount of specific antibodies in the serum. When testing a group of rabbit sera, negative for EIA virus antibodies by the AGID test, in the SPRIA a range of positive reactivities was noted. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody.     

Monoclonal antibodies to Marburg virus proteins and their immunochemical characteristics]

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus proteins are prepared. Positive hybridomas were selected by solid-phase enzyme immunoassay (EIA) by specificity of their immunoglobulin reaction with Marburg virus antigen. Virus proteins reacting with MAbs were identified by immunoblotting. Out of 20 examined hybridoma antibodies, 5 reacted with protein VP35, 5 with VP40, 3 with NP, 1 with protein complex VP35-VP40, MAb 7H10 detected 2 proteins (VP40 and NP), and 5 MAbs did not bind virus proteins in this assay. Marburg virus antigen adsorbed on the surface of plates were detected by indirect EIA with biotin-treated MAbs (PEIA-MAb) and indirect EIA (IEIA-MAb). The sensitivity of both methods differed with different hybridoma antibodies and was the maximum with MAb 5F1 specific to Marburg virus nucleoprotein: 5-10 and 1-2 ng/ml for the direct and indirect methods, respectively. Purified MAbs 7C4, 7D8, and 5F1 were used as antigen captures in EIA for detecting immunoglobulins to Marburg virus in a serum from convalescent after Marburg fever. The results recommend the above MAbs for use in test systems for the diagnosis of the disease and detecting virus antigen.     

Expression of a gene encoding the major antigenic protein 2 homolog of ehrlichia chaffeensis and potential application for serodiagnosis

The major antigenic protein 2 (MAP2) homolog of Ehrlichia chaffeensis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody. However, immune sera failed to react with the recombinant on immunoblots when the antigen was denatured by heat or reduced using beta-mercaptoethanol. The recombinant MAP2 (rMAP2) was used in an ELISA format with 60 blinded serum samples. Twenty of the serum samples were previously demonstrated to contain antibodies reactive with E. chaffeensis by indirect immunofluorescence assays (IFAs). The remaining 40 samples were seronegative. All samples negative by IFA were also found to be negative for antibodies against the rMAP2 of E. chaffeensis by using the ELISA. Only 1 of 20 IFA-positive samples tested negative in the rMAP2 ELISA. There was 100% agreement using IFA-negative samples and 95% agreement using IFA-positive samples, resulting in a 97.5% overall agreement between the two assays. These data suggest that the rMAP2 homolog of E. chaffeensis may have potential as a test antigen for the serodiagnosis of human monocytic ehrlichiosis. To our knowledge, this recombinant is unique because it is thus far the only E. chaffeensis recombinant antigen that has been shown to work in an ELISA format.     

猪圆环病毒2型Cap基因的原核表达及ELISA检测方法的建立

目的:方法:为建立PCV-2 ELISA检测方法.利用PCR技术从PCV-2基因组中克隆Cap抗原表位基因,并将其插入到载体pET-22b(+)中,构建成原核表达质粒pET-22b(+)-Cap,使其在E.coli BL21(DE3)中以IPTG诱导表达并进行纯化,以表达的蛋白进行包被,建立ELISA检测方法.结果:表达产物分子质量约30 kD,经Western blot表明具有良好的免疫活性,对643份猪血清进行检测,阳性率为73.1%,阴性率为26.9%.结论:该检测方法能在临床中进行应用.     

Detection of specific antibodies toToxoplasma gondii by a competitive enzyme immunoassay using a monoclonal antibody against the P30 antigen

An inhibition EIA using a monoclonal antibody against the major P30Toxoplasma gondii surface protein was designed for detection of specific antibodies in human sera. The assay was based on the inhibition of binding of peroxidase labelled monoclonal antibody toToxoplasma gondii crude antigen coated plates by the corresponding antibodies present in human sera. This rapid and simple assay was compared to indirect immunofluorescence, direct agglutination and an immunosorbent agglutination assay using 435 human sera. The specificity and sensitivity were 100 % and 97 % respectively. This test was found to be as sensitive as the dye test.     

Enzyme immuno assay for the detection of virus specific IgG and IgM antibody in patients with Haemorrhagic Fever with Renal Syndrome

Summary Consecutive serum samples collected from 235 patients with Haemorrhagic Fever with Renal Syndrome (HFRS), between two days and two years after onset of disease, have been analysed for the presence of IgG and IgM type of antibodies specific for Hanta-viruses. The sera were screened in parallel by a newly developed indirect Immuno Enzyme Assay (EIA) in parallel with Indirect Immunofluorescent Antibody Assay (IFA). In both tests the Hantaan virus strain 76–118 was used as the antigen. The EIA was much more sensitive than the IFA test for the detection of IgM type antibodies. With the indirect EIA IgM type antibodies against Hantaan virus 76–118 have been detected in HFRS patient's sera from the second day of illness indicating the usefulness of this test for the early serological diagnosis of this disease.     

Development of a highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses enzyme-labeled antigen.

An enzyme immunoassay (EIA) for serum immunoglobulin M (IgM) antibodies to rubella virus based on enzyme labeling of viral antigen was developed. The sensitivity of the EIA for the detection of recent rubella virus infection was evaluated by using 115 rubella-IgM-antibody-positive serum specimens, which were confirmed as positive by Rubazyme M (Abbott Diagnostics). In addition, 12 individuals, 2 of whom were exposed to rubella through vaccination and 10 of whom were exposed through natural infection, were studied, and the results were compared with those obtained by indirect EIA (Rubelisa M; Electro-Nucleonics, Inc.) and immunoblotting. The sensitivity of the newly developed EIA with sera from these individuals was 100%. Serum specimens from two patients indicated that the IgM antibodies were detected by the newly developed EIA at the same time as IgM antibodies were detected by immunoblotting and before positive reactions were detected by an indirect EIA. The reference population consisted of 564 healthy blood donors and hospitalized patients (150 serum specimens). In addition, 145 serum specimens commonly giving false-positive reactions in conventional rubella IgM EIAs were studied. With these specimens, no false-positive reactions were observed. Positive IgM responses, which could not be confirmed by immunoblotting, were observed in two samples from the reference population. However, these two samples were rubella IgG positive. The overall specificity of the EIA was 99.8%.     

Human immune response against outer membrane proteins of Moraxella (Branhamella) catarrhalis determined by immunoblotting and enzyme immunoassay.

The role of Moraxella (Branhamella) catarrhalis as a respiratory tract pathogen is increasingly recognized. We looked at the human immune response against individual outer membrane proteins of M. catarrhalis and against the 81-kDa CopB protein, which has previously been shown to be a target for protective antibodies. Paired serum samples from six elderly patients with pneumonia were tested by Western blot (immunoblot) analysis by using outer membrane vesicles of M. catarrhalis 035E as antigen. All of the six convalescent-phase serum samples reacted with a protein which migrated at the position of the CopB protein and with a high-molecular-weight protein of M. catarrhalis; three serum samples also reacted with a 34-kDa outer membrane protein. Paired serum samples from 18 patients, 10 of which had M. catarrhalis infection on the basis of previous serology results, were tested by enzyme immunoassay (EIA) with the CopB protein and whole cells of M. catarrhalis 035E as antigens. Nine patients showed a significant rise in EIA titer between acute- and convalescent-phase sera when whole bacterial cells were used as antigens. Six (67%) patient samples that were positive by the EIA with the whole-cell antigen were also positive by the EIA with the CopB antigen, and six of nine patient samples negative by the EIA with the whole-cell antigen were also negative by the EIA with the CopB antigen. These results suggest that both the CopB and a high-molecular-weight protein are major targets of the immune response against M. catarrhalis, and further studies with greater amounts of patient materials are needed to elucidate the usefulness of CopB as an antigen in etiologic studies.     

Genetic diversity of Brazilian strains of porcine circovirus type 2 (PCV-2) revealed by analysis of the cap gene (ORF-2)

Summary Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, 19 of 870 samples from pigs from different Brazilian states were found to be positive for PCV-2 by polymerase chain reaction (PCR). A fragment of 700 nt of the cap gene (ORF-2) from the 19 PCV-2-positive samples were sequenced using three pairs of primers (Fa/Ra, Fb/Rb and Fc/Rc). Maximum parsimony genealogy with a heuristic algorithm using the 19 field strain studied here, 21 sequences from GenBank and PCV-1 as an out-group showed the existence of two major clusters (A and B) and the Brazilian strains segregating in both of them. PCV-2 was found in pigs with various clinical signs. No association between clusters of PCV-2 and different states or clinical signs were observed, demonstrating that the exact role of PCV-2 in porcine circovirus diseases (PCVD) in Brazil still needs to be clarified. These results contribute to the molecular characterization of PCV-2, which serve as a basis for the epimiology of PCV-2 infection.     

Evaluation of immunoassays for detection of antibodies to human herpesvirus 7.

An enzyme immunoassay (EIA), an immunoblot assay (IB), and an indirect immunofluorescence assay were developed for detection of human herpesvirus 7 (HHV-7) antibodies in human serum. Cross-absorption studies with EIA or IFA using HHV-7 and human herpesvirus 6 (HHV-6) antigens indicated that most human sera contain cross-reactive HHV-6 and HHV-7 antibodies and that the degree of cross-reactivity varies between individual serum specimens. Inhibition of homologous antibody activity by absorption with heterologous virus ranged from 0 to 57% by EIA. However, for every sample tested, absorption with homologous virus removed more activity than did heterologous virus. An 89-kDa protein was identified as an HHV-7-specific serologic marker by IB. Activity to this protein was not removed by absorption with HHV-6 antigen. Of the three assays, the EIA was the most sensitive (94%), while the IB was the most specific (94%). Approximately 80% of specimens collected from German adults and children older than 2 years were positive for HHV-7 antibodies by these assays.     

Expression of the C-terminal ORF2 protein of duck astrovirus for application in a serological test

Duck astrovirus (DAstV) is an important pathogen causing duck viral hepatitis (DVH), a highly contagious and fatal disease in young ducklings. To provide an antigen for a diagnostic serum test, the C-terminus of DAstV ORF2 protein was expressed in Escherichia coli. Four positive and 30 negative sera were used to validate the purified ORF2 protein by developing an indirect enzyme-linked immunosorbent assay (ELISA). No cross-reactions were found against other duck pathogens, including duck hepatitis A virus, duck plague herpesvirus, duck reovirus, Newcastle disease virus, and Riemerella anatipestifer 12/19 (63.2%) and 26/51 (51%) sera samples from two flocks of ducks that survived DAstV infections in commercial duck farms were positive for DAstV by this method, respectively. Interestingly, DAstV-specific antibodies were also detected in 12 (28.6%) of 42 sera samples from a different flock without DVH, indicating a wide distribution of subclinical infections caused by DAstV.     

Detection of HEV antigen as a novel marker for the diagnosis of hepatitis E

Infection with hepatitis E virus (HEV) may be diagnosed by the presence of HEV RNA or anti-HEV antibodies. An enzyme immunoassay (EIA) was developed for the detection of antigen. Twenty-four monoclonal antibodies (mAbs) were produced. An indirect sandwich EIA was developed to detect HEV antigen using a combination of three mAbs as coating antibodies. Approximately 44.6% (33/74), 28.6% (50/175), and none (0/27) of sera positive for anti-HEV IgM alone, both anti-HEV IgM and IgG, and anti-HEV IgG alone also were positive for HEV antigen using this EIA. Forty-two HEV antibody-positive sera were tested for HEV RNA and antigen in parallel and the concordance was 81.0% (34/42). All PCR products were found to belong to HEV genotype 4. In order to evaluate the temporal relationship between HEV antigen positivity and HEV RNA, anti-HEV IgG and IgM, and ALT concentrations, macaques were infected with HEV genotypes 1 and 4 and serial samples were collected. The results showed that the antigen EIA can detect the capsid proteins of both genotypes. HEV antigen was detectable prior to ALT elevation and the appearance of anti-HEV antibodies in the infected monkeys and lasted for several weeks in all cases. HEV antigen became detectable in the serum at almost the same time as HEV RNA in feces but persisted for 4 weeks less than HEV RNA. This assay should be valuable for the diagnosis of acute hepatitis E, particularly in the window period prior to seroconversion to anti-HEV.     

Production and application of new monoclonal antibodies specific for a fecal Helicobacter pylori antigen.

The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.     

Immunoenzyme analysis of monoclonal antibodies on solid-phase infected cells

Monoclonal MAK-14-7 antibodies to the surface antigen of Venezuelan equine encephalomyelitis virus and OKA series to vaccinia virus antigens were studied by enzyme-immunoassay (EIA) on the solid phase of the infected Vero, BHK-21, and HeLa cells. The immunoglobulins under study from the culture and ascitic fluids of hybridomas were shown to bind specifically with the appropriate antigens of the infected cells. The activity of monoclonal antibodies to viral antigens in EIA on the solid phase of the infected cells was 10-fold higher than in indirect immunofluorescence test. The method has a number of useful advantages such as the simplicity of preparation of the antigen solid phase, rapidly obtainable results, long-term preservation of ready panels with the specific antigen at room temperature. The high sensitivity of the method makes it effectively useful for screening of hybrid clones producing monoclonal antibodies.     

Assessment of a recombinant antigen versus natural hypodermin C for the serodiagnosis of hypodermosis in cattle

An indirect ELISA test using as antigen a recombinant parasite protein, hypodermin C, was developed to measure Hypoderma-specific antibodies in cattle sera and compared with natural hypodermin C. To evaluate the field efficacy of the ELISA test, 334 serum samples were collected from cows raised at farms in Galicia for a serological survey. Compared with an ELISA based on natural parasite antigen, the recombinant hypodermin C gave excellent results, with a sensitivity of 95.8% and a specificity of 95.7%. Considering the cut-off point, with the recombinant hypodermin C, 70.9% of the animals had positive levels of antibodies to Hypoderma and with natural hypodermin C, 73.6%. Recombinant hypodermin C appears to be a useful alternative to the natural parasite antigen for the serodiagnosis of Hypoderma sp in cattle. Received: 26 March 1999 / Accepted: 21 April 1999     

Large-Scale Evaluation of the Immuno-Mycologics Lateral Flow and Enzyme-Linked Immunoassays for Detection of Cryptococcal Antigen in Serum and Cerebrospinal Fluid

Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results.     

Serodiagnosis ofHelicobacter pylori infections with an enzyme immunoassay using the chromatographically purified 120 kilodalton protein

A membrane-associated 120 kDa protein ofHelicobacter pylori with known species-specificity was isolated and used in an enzyme immunoassay (EIA) for the detection ofHelicobacter pylori-specific IgG antibodies in patient sera. The EIA was compared with two other methods used for serodiagnosis ofHelicobacter pylori infections: an EIA using sonicated wholeHelicobacter pylori cell antigen and Western immunoblot. In a prospective study 127 unselected patients (76 patients with antrum gastritis, 51 patients without gastritis) who underwent gastroscopy were studied histologically and serologically. The EIA using the purified 120 kDa protein had the highest specificity (92 %) compared with the EIA using a whole cell sonicate of a singleHelicobacter pylori strain as antigen (60.7 %) and the immunoblot (90.2 %). The sensitivity was 96 %, 100 % and 92 %, respectively. Sera of three control patients reacted strongly in all three methods, indicating possibleHelicobacter pylori infection with negative histological findings. The EIA using the 120 kDa protein as antigen was shown to be a specific and sensitive technique for the serodiagnosis ofHelicobacter pylori infections.