Serodiagnostic comparison of enzyme-linked immunosorbent assay and surface plasmon resonance for the detection of antibody to Porcine circovirus type 2

This paper describes the cloning and expression of the capsid protein of Porcine circovirus type 2 (PCV2) in an Escherichia coli expression system that was used to produce a fusion protein for subsequent immunologic studies: enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Polymerase chain reaction was used to amplify the gene encoding the capsid protein from the DNA of PCV2. The protein was then cloned into a pRSET prokaryotic expression vector. Western blot analysis revealed that the recombinant protein gave strong signals on a polyvinylidene difluoride membrane when exposed to the serum from a pig infected with PCV2. The expressed protein was purified and used as an antigen for the ELISA and SPR study. A protein chip based on SPR was developed, and the diagnostic potential of SPR was compared with that of ELISA with the use of 70 serum samples obtained from 6 pig farms. There was a strong positive correlation between the ELISA and SPR titers (r = 0.877, P < 0.01). Therefore, this recombinant capsid protein can be used as an antigen for serologic studies, and the SPR, a label-free method, appears to be a valuable and reproducible tool in the serodiagnosis of a PCV2 infection.     

Serodiagnostic comparison between two methods, ELISA and surface plasmon resonance for the detection of antibodies of classical swine fever

A protein chip based on surface plasmon resonance (SPR) was developed to measure the antibody (Ab) titers of classical swine fever virus (CSFV) using the recombinant gp55 protein as an antigen. The diagnostic potential of this SPR assay for detecting the Ab titers to CSFV gp55 was compared that of the enzyme-linked immunosorbent assay (ELISA) using 170 serum samples from 14 pig farms. The SPR assay was highly specific and sensitive, and there were no cross-reactions detected. There was a strong positive correlation between the SPR and ELISA titers (n=170, r=0.869, p<0.01). Therefore, the SPR label-free method is a valuable tool in the serodiagnosis of CSFV infection and determining Ab titers after vaccination.     

猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立

猪肺炎支原体是猪喘气病的病原体,本研究选择猪肺炎支原体P46膜蛋白基因亲水区序列进行克隆,并将其内3个编码Trp的TGA突变成TGG,然后再克隆到pET28a(+)载体中,在大肠杆菌BL21 (DE3)细胞内实现了高效表达,表达产物相对分子质量约为31 ku,约占菌体总蛋白35％,表达形式为包涵体,通过Western blotting 证明表达产物与猪肺炎支原体高免血清具有很好的反应原性和特异性.将大肠杆菌表达的猪肺炎支原体P46重组蛋白经过洗涤、过柱纯化后,作为间接ELISA包被抗原用于检测猪血清中猪肺炎支原体抗体,通过对各参数和试剂的优化建立了rP46-ELISA方法,获得了较好的效果,通过与现有ELISA检测方法的比较,结果表明二者间具有较高的符合率.     

In vitro expression of the 50-kDa and 30-kDa fragments of the P97 adhesin of Mycoplasma hyopneumoniae in Escherichia coli and their use for serodiagnosis

This article reports the cloning and expression of 2 fragments of the P97 adhesin of Mycoplasma hyopneumoniae for use in serodiagnosis: a 50-kDa fragment (including the N-terminal cleavage site) and a 30-kDa fragment (including the C-terminal R1 and R2 repeats, which are essential for adherence). The genes encoding the fragments were amplified, cloned, and expressed in the Escherichia coli expression system BL21 (DE3)pLysS. Antiserum against the purified recombinant proteins reacted with the mycoplasmal 97-kDa intact protein and the 66-kDa major cleavage fragment, confirming that both cloned fragments could induce antigen-specific antibodies in mice. Of 70 serum samples from nonvaccinated pigs, 26 (37%) were seropositive when the 30-kDa fragment was used as an antigen for enzyme-linked immunosorbent assay, suggesting that natural mycoplasmal infection is quite common in Korea. However, only 4 samples were seropositive when the 50-kDa fragment was used; this fragment was therefore deemed unsuitable for serodiagnosis. The 30-kDa fragment protein might be useful for measuring antibody response to vaccination and for detecting mycoplasmal infection.     

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.     

Comparison of surface plasmon resonance imaging and enzyme-linked immunosorbent assay for the detection of antibodies against iridovirus in rock bream (Oplegnathus fasciatus).

A protein chip based on surface plasmon resonance imaging (SPRI) was developed for detecting fish iridovirus antibody using a recombinant 50-kDa fragment of major capsid protein (MCP) as an antigen. The diagnostic potential of SPRI for measuring antibodies to the iridovirus MCP was compared with that of a conventional enzyme-linked immunosorbent assay (ELISA) using 40 juvenile rock bream (Oplegnathus fasciatus) serum samples in a nursery. There was a strong positive correlation between the SPRI and ELISA (n = 40, r = 0.939, P < 0.01). Therefore, this recombinant 50-kDa MCP can be used as an antigen for serological studies, and the SPRI, which is a label-free and high-throughput method, is potentially a valuable tool in the serodiagnosis of an iridoviral infection.     

In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells.

A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigen-reactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.     

猪肺炎支原体乳酸脱氢酶基因的克隆、表达、纯化及其间接ELISA检测方法的建立

根据GenBank中的猪肺炎支原体乳酸脱氢酶（LDH）基因序列设计1对引物，PCR扩增LDH基因，首先将扩增片段连接到克隆载体pMD18-T上，然后连接到表达载体pGEX—KG上，经测序正确后诱导表达。重组质粒在大肠杆菌中表达的目的蛋白以可溶性蛋白和包涵体2种形式存在。将超声波破碎的诱导菌液高速离心，其上清用Glutathione Sepharose4Bbead亲和层析纯化。用猪肺炎支原体的标准阳性血清对纯化蛋白作Western—blot检测，出现目的条带；以纯化蛋白为抗原建立了检测猪肺炎支原体抗体的间接ELISA方法，该方法具有较好的稳定性和重复性，较高的特异性与敏感性。用建立的ELISA方法与中国兽医药品监察所研制的IHA试荆盒同时检测120份临床血清，二者总符合率为92．5％。用建立的ELISA方法检测了671份临床送检不同年龄阶段的猪血清，结果显示断奶前仔猪猪肺炎支原体（Mycoplasma hyopnenmoniae，MHP）感染率为44．3％，保育猪为3．0％，育肥猪为17．44％，种猪为73．41％，这初步反映了猪喘气病在各个年龄阶段的感染率。     

Evaluation of an enzyme-linked immunosorbent assay for the detection of Mycoplasma hyopneumoniae antibody in porcine serum

An enzyme-linked immunosorbent assay (ELISA) for detecting antibody to Mycoplasma hyopneumoniae in porcine serum is described. The results are presented as an ELISA ratio, calculated by dividing the absorbance of the test sample by the mean absorbance of control negative sera. In known infected pigs, the ELISA ratio was highest when the serum concentration applied to the ELISA plate was diluted 1 in 20 in PBS - Tween. Mean ELISA ratios ranged from 1.2 +/- 0.3 for pigs without porcine enzootic pneumonia (PEP) lesions to 5.5 +/- 1.5 for pigs observed with a PEP lesion reacting positively with immunofluorescent histopathology. Pigs observed with typical PEP lesions at slaughter, but not confirmed by immunofluorescent histopathology had a mean ELISA ratio of 4.9 +/- 1.7. The ELISA was highly sensitive (95.6%) and specific (98.8%) when pig sera from commercial piggeries of known M hyopneumoniae infection status were assessed. No cross-reactivity with serum from a pig hyperimmunised with killed M flocculare was detected, and reactivity with serum from another pig hyperimmunised with killed M hyorhinis showed only weak cross-reactivity, which failed to reach the ELISA positive threshold (ELISA ratio 3) for M hyopneumoniae.     

Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis

A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation can be derived that allows demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).     

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.     

Evaluation of the indirect hemagglutination assay as a practical serodiagnostic test for mycoplasmal pneumonia of swine

Sera from swine experimentally or naturally infected with Mycoplasma hyopneumoniae (the etiological agent of mycoplasmal pneumonia of swine, MPS) were tested by the indirect hemagglutination assay (IHA), the enzyme-linked immunosorbent assay (ELISA) and the complement fixation (CF) test. The IHA detected antibody at comparable times and levels to the other 2 serological tests following experimentally-induced infection. In the late antibody response (greater than or equal to 86 days post-infection), the ELISA titres were higher than either the IHA or the CF test. The IHA appeared least satisfactory when it was used to test sera from commercial swine herds. When 1000 sera were tested, the IHA was positive for only 30 (22%) of 135 sera which were positive by the ELISA and the CF test. The IHA titres were low; 20 of the 30 sera had a titre of only 10. The end-points for the IHA were difficult to read for sera of this low titre. The relationship between positive IHA results for the herd sera obtained at necropsy, and the occurrence of gross or microscopic lesions typical of MPS was poor (41 and 50% agreement, respectively). An agreement of 39% was noted between positive IHA results and the localization of mycoplasmal antigens by an indirect immunofluorescence (IIF) test. However, IHA results correlated significantly (P less than 0.05) with gross and microscopic lesions, but not with the IIF test. No significant correlation was noted between the IHA (or the other 2 serologic tests) and the cultural isolation of M. hyopneumoniae or M. flocculare. On the basis of these results, the IHA appears to have limited promise as a practical test for the diagnosis of MPS in commercial swine herds because of the low titres observed, poor correlation of the IHA and other indicators of MPS, the necessarily subjective determination of end-points, and other inherent technical limitations of the test.     

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.     

Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples.

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.     

A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and reproducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.     

Serodiagnosis of canine leptospirosis by solid-phase enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Leptospira interrogans serotype canicola in dogs was developed and evaluated. Comparison of the ELISA with the microscopic agglutination test (MAT) showed that, during the first two weeks after an experimental infection with serotype canicola, the ELISA detected antibody at higher dilutions than the MAT. After the second week post-infection both tests detected antibody at almost equal titres (r = 0.89). The outer envelope (OE) antigen of serotypes icterohaemorrhagiae, copenhageni and canicola was fairly serotype-specific, whereas the pellet (P) antigen showed more cross-reactivity. Both OE and P antigen of Leptospira biflexa strain Patoc I could be used as cross-reacting antigen in the ELISA. Compared to the MAT, the ELISA has some technical advantages. It is suggested that the ELISA would be useful as a screening test.     

小反刍兽疫病毒核衣壳蛋白抗原性与特异性的比较

为筛选建立小反刍兽疫病毒N蛋白双抗原夹心ELISA抗体检测方法的抗原原料,本研究通过优化大肠杆菌密码子、优化蛋白表达与纯化条件等方法,分别在pET-30a与pET-32a两个不同原核表达载体中成功获得了可溶性的小反刍兽疫核衣壳蛋白(N)。分别对重组大肠埃希氏菌pET-30a-(N)-BL21(DE3)株与pET-32a-(N)-BL21(DE3)株种子批的P7代与P20代进行蛋白诱导表达与抗原提取纯化,共获得6批小反刍兽疫病毒pET-30a-(N)蛋白与pET-32a-(N)蛋白,并对蛋白的浓度、纯度、抗原性和特异性进行检验。结果表明两个纯化后的重组抗原与羊小反刍兽疫病毒阳性血清有明显反应,具有较好的反应性,而与羊痘病毒阳性血清、羊口蹄疫病毒阳性血清、山羊关节炎/脑炎病毒阳性血清等特异性血清均无交叉反应,具有较好的特异性。本研究结果为今后以N蛋白为基础建立相应的抗原捕获ELISA方法,竞争ELISA检测方法、间接ELISA检测方法以及双抗原夹心ELISA抗体检测方法打下了坚实的基础。     

Antibody response in salmonids against the 70 kDa serine protease of Aeromonas salmonicida studied by a monoclonal antibody-based ELISA

An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida. This new assay permits a specific determination of anti-protease-antibodies, without antigen purification. Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies. Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations (r=0.753). Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease. A conjugate of inhibited protease and cationized bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species (P<0.05), whereas the unconjugated antigen and Apoject 1-Fural were proved to be ineffective. Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity. The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinize its protective significance.     

Serum and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant

Objective To investigate the protective efficacy of a pool of denatured membrane protein antigens of Mycoplasma hyopneumoniae (J strain) in the molecular size range 70 to 85 kDa (F3 antigen) in combination with adjuvants for pigs challenged with M hyopneumoniae .
Design A vaccine efficacy experiment with assessment of serum and respiratory tract antibody responses.
Procedure F3 antigens were emulsified with five different adjuvants. To groups of three pigs per vaccine, four vaccines were given by intramuscular injection, and two vaccines, including one of those given intramuscularly, were given by intraperitoneal injection.
Results Compared to six unvaccinated pigs, animals vaccinated with F3 antigen displayed significantly reduced pneumonia (54% reduction in mean lung score) following experimental challenge. Analysis of post-vaccination, pre-challenge IgG and IgA ELISA antibody absorbances in serum and respiratory tract washings revealed no correlation with lung score. Six weeks after challenge, pigs previously vaccinated intramuscularly mostly demonstrated greater IgG and IgA responses in respiratory tract washings, and greater IgG serum antibody responses, than those vaccinated by intraperitoneal injection.
Conclusion Pigs vaccinated with M hyopneumoniae antigens in the molecular size range of 70 to 85 kDa showed a significant reduction in lung lesions compared with unvaccinated control animals after experimental challenge. IgG and IgA antibody concentrations in serum and respiratory tract washings after vaccination do not provide a useful prognostic indicator of protection from enzootic pneumonia.     

Comparison of complement fixation test and enzyme-linked immunosorbent assay for detection of early infection with Mycoplasma hyopneumoniae

The relative merits of the complement-fixation test (CF) and enzyme-linked immunosorbent assay (ELISA) for the detection of the early antibody response to Mycoplasma hyopneumoniae were evaluated. Discriminant analysis, a statistical procedure, was used to avoid difficulties associated with variation in background color and nonspecific reactions obtained with ELISA with different sera. Specific-pathogen-free pigs were exposed by contact to other specific-pathogen-free pigs which had been inoculated with M hyopneumoniae intratracheally (experiment A) or intranasally (experiment B) 18 to 21 days previously. Sera were collected from each pig before contact exposure and once a week until necropsy. Antibodies were detected by CF at postexposure (PE) week 3 in animals in experiment A (6 of 18) and at PE week 5 in experiment B (3 of 12). The ELISA antibodies were detected at 2 weeks after beginning of contact exposure in experiments A (4 of 18) and B (1 of 12). Examination of pooled data for experiments A and B indicated that ELISA was substantially (P less than 0.05) more sensitive for detection of antibodies than was the CF test at 3 to 5 weeks after contact exposure began. At PE weeks 6 and 7, both tests were similarly effective in detecting M hyopneumoniae antibodies.