Serodiagnostic comparison between two methods, ELISA and surface plasmon resonance for the detection of antibody titres of Mycoplasma hyopneumoniae

A protein chip based on surface plasmon resonance (SPR) was developed for measuring the Mycoplasma hyopneumoniae antibody titres using a recombinant 30-kDa fragment of P97 adhesin as an antigen. The diagnostic potential of this SPR assay, for detecting the antibody titres to the M. hyopneumoniae 30-kDa protein, was compared with that of conventional ELISA using 70 pig serum samples taken from six pig farms. The SPR assay was found to be highly specific and sensitive. Moreover, there was a strong positive correlation between the SPR and ELISA titres (n = 70, r = 0.898, P < 0.01). Therefore, this recombinant 30-kDa protein can be used as an antigen for serological studies, and the SPR, which is a label-free method, is expected to be a valuable and reproducible tool in the serodiagnosis of M. hyopneumoniae infection.     

Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2.

We report the development of a competitive enzyme-linked immunosorbent assay (c-ELISA) for the detection of antibodies to porcine circovirus type 2 (PCV2), the agent associated with the recently described postweaning multisystemic wasting syndrome in pigs. At present, no method has been published describing a c-ELISA for the detection of antibodies to PCV2, and currently employed tests are impractical for use in some laboratories. The assay described here uses a cell culture isolate of porcine circovirus type 2 as antigen and a PCV2-specific monoclonal antibody as the competing reagent. Evaluation of the ELISA was performed by comparison with results obtained using an indirect immunofluorescent test on 484 sera from pig herds in the United Kingdom, Canada, France, and the USA and serial bleeds from pigs experimentally infected with porcine circoviruses. The sensitivity and specificity of the ELISA were determined as 99.58% and 97.14%, respectively, at 2 standard deviations (SD) from the mean or 95.81% and 100% at 3 SD from the mean. Using this ELISA, a serologic survey of 461 sera collected from commercial pig herds in Northern Ireland between 1973 and 1999 was undertaken. Analysis of the results of this survey demonstrated that the number of ELISA-positive sera detected in an individual year during this period ranged from 55% to 100%. This c-ELISA has applications for large-scale rapid diagnosis of PCV2 infection in pig populations worldwide and for immunoscreening of sera from other species for antibodies to PCV2.     

猪捷申病毒与猪圆环病毒2型双重PCR检测方法的建立及初步应用

为建立可同时检测猪捷申病毒（PTV）与猪圆环病毒2型（PCV2）的双重PCR方法,本研究根据GenBank登录的相关病毒基因序列,选择保守区域设计引物,经过反应条件的优化,建立了可同时检测以上2种病毒的PCR诊断方法,扩增片段长度分别为187bp（PTV）、120bp（PCV2）。通过实验证明该方法具有良好的特异性和较高的敏感性,对PTV、PCV2核酸检测最低浓度分别为2．8×10^-2ng和6．6×10^-3ng。应用该方法对43份临床样品进行检测发现,PTV阳性率为23％,PCV2阳性率为38％,PTV与PCV2共感染率为16％。该方法的成功建立,为快速高效地检测以上2种病毒提供有力手段。     

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.     

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.     

An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to type A avian influenza (AI) virus. The sensitivity and group specificity of the AI-ELISA were compared with those of the agar-gel-precipitin test (AGPT) and the hemagglutination-inhibition (HI) test under conditions of both controlled and field exposure. During the course of temporal experimental infection (0-76 days) of specific-pathogen-free (SPF) chickens with AI subtype Hav9N2, the AI-ELISA was able to detect specific AI antibody as early as 8 days postinoculation (PI), and it measured rising levels of antibody through 35 days PI, at which time the chickens were re-exposed to AI virus. Conversely, AGP tests were negative through 35 days PI, and HI tests began to detect low levels of AI antibody only at 21 days PI. Following a secondary infection at 35 days PI with the same AI subtype, all tests measured rising levels of AI-specific antibody (35-76 days PI). However, the AGP test was positive at only the 7- and 14-day samplings postsecondary immunization. Under field conditions, the AI-ELISA was able to detect serum AI antibody in flocks from which highly pathogenic AI was isolated, but the AGP tests of these sera were negative.     

Porcine circovirus type 2 antibody detection in backyard pigs from Mexico City

PCV2 antibodies have been found in pigs from all continents. However, this finding has been mainly studied in domestic swine reared under intensive production conditions. Mexico City, with a human population over 19 million in 2005, has both urban and rural areas. The pig production in its rural area is based on small family backyard farms. Taking into account this rather unique form of rearing pigs, the objective of this study was to determine the seroprevalence in backyard pigs from the rural area of Mexico City. A total of 695 backyard pig serum samples from 108 small family farms belonging to seven municipal areas were studied by immunoperoxidase monolayer assay technique. One hundred six out of the 108 family farms (98.14%) had at least one positive serum sample. On the other hand, 136 (19.57%), 264 (37.99%) and 248 (34.82%) pigs had low, intermediate and high titres to PCV2, respectively. Only 53 samples (7.63%) were negative for PCV2 antibodies. No apparent differences in antibody titre groups were observed among backyard pigs from the different municipal areas. In conclusion, the present study, the first one performed in this kind of extensively produced pigs, indicates that PCV2 is ubiquitous in backyard pigs from Mexico City.     

An enzyme-linked immunosorbent assay for the detection of Clostridium perfringens enterotoxin antibody.

An enzyme-linked immunosorbent assay (ELISA) was adapted to test serum antibody to enterotoxin of Clostridium perfringens type A. The test was evaluated using sheep, calf and guinea pig sera and compared with passive hemagglutination and immunodiffusion tests. The ELISA was found to be more sensitive than the other two tests and was completely free from nonspecific reactions. The method was considered to be technically advantageous and suitable for semiautomated procedures.     

H5 antibody detection by blocking enzyme-linked immunosorbent assay using a monoclonal antibody

Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody to Pasteurella haemolytica: constructing an enzyme-linked immunosorbent assay titer

A 2-stage strategy was developed and evaluated for estimating serum antibody titer by use of ELISA and a series of dilutions. In stage 1, the linear response region and least-square estimate of the assay line slope were established from 9-point dilution assays. Provided that the reading was within the linear response region, this information was used in the stage-2 estimation of titer from a single absorbance reading. Operationally, 2 fixed dilutions were selected, one suitably low and one suitably high, to provide at least one reading within the linear region. The procedure should save considerable time when a large number of assays are to be performed. Stage 1 required approximately twenty 9-point assays, but all subsequent assays required only 2 fixed dilutions.     

一种猪圆环病毒Ⅱ型ELISA检测试剂盒的临床应用

本研究通过采用猪圆环病毒2-dCap-ELISA抗体检测试剂盒,对河北、山西两地共441份猪血清样品进行检测发现：河北保定地区349份血清样品中阳性样品为306份,感染率为87.68%;山西地区92份血清样品中阳性样品为77份,感染率为83.7%。与标准的间接免疫荧光检测法进行比较,发现相对于标准IFA,该ELISA检测试剂盒的诊断敏感性为95.48%(383/398),诊断特异性为93.02%(40/43),总符合率为95.24%。由此表明该试剂盒是一种简便、快速、灵敏、适于大规模临床应用的猪圆环病毒2型抗体检测试剂盒。     

Studies on the detection of antibody to duck hepatitis virus by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to duck hepatitis virus (DHV) is described. The results of ELISA were compared with those of an agar gel diffusion precipitin (AGDP) test and a serum-neutralization (SN) test. The specificity of the ELISA was in accordance with the specificity of the AGDP and SN tests, but there was a difference in sensitivity. The positive detection rates of ELISA, SN test, and AGDP test for 93 clinical samples were 68.8%, 68.8%, and 18.8%, respectively. A positive/negative (P/N) value larger than or equal to 2.1 plus an absorbance value larger than or equal to 0.4 was used as a comprehensive positive standard for the ELISA. This eliminated false-positive reactions. The results showed that the ELISA was a rapid, sensitive, and accurate method for detecting antibody to DHV.     

Sandwich enzyme-linked immunosorbent assay by using monoclonal antibody for detection of Clostridium perfringens enterotoxin

Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative estimation of Clostridium perfringens enterotoxin (CPE) with monoclonal and polyclonal antibodies as capturing and detecting antibodies, respectively. The dose-dependent relationship between absorbance at 405 nm and concentration of purified CPE was obtained over the range of 0.64-400 ng/ml. The sandwich ELISA was fond to detect crude CPE in culture and CPE in 10% fecal extracts. This method is convenient, rapid and sensitive for specific detection of CPE.     

A double-antibody enzyme-linked immunosorbent assay for the detection of turkey hemorrhagic enteritis virus antibody and antigen

A highly sensitive and specific double-antibody enzyme-linked immunosorbent assay (ELISA) is described for the detection of antigen and antibody of turkey hemorrhagic enteritis virus (HEV). The assay utilizes a virus-neutralizing monoclonal antibody (MAb) to capture the antigen and turkey antiserum against HEV as the second antibody. Microtiter plates were first coated with a dilution of 1:3000 of the MAb (300 ng immunoglobulin/well) and are used for detection of both antigen and antibody. For antibody detection, MAb-coated plates were treated with an appropriate dilution of a cell-culture-propagated HEV antigen and then reacted with the test turkey serum. For detection of HEV antigen, MAb-coated plates were treated with appropriate dilutions of test antigens and then reacted with purified anti-HEV turkey immunoglobulins. The assay for HEV antibody detection was more sensitive and specific than previously described single-antibody ELISAs. Using the double-antibody ELISA, it was found that the spleen of HEV-infected turkeys harbors very high levels of antigen. Traces of HEV antigen are present in some other organs. Infectivity assay for HEV is found to be about two orders of magnitude more sensitive than the ELISA for detection of virus.     

The relationship between the hemagglutination-inhibition test and the enzyme-linked immunosorbent assay for the detection of antibody to Newcastle disease

An assay of 364 chicken serum samples for Newcastle disease virus antibodies determined that a commercial NDV enzyme-linked immunosorbent assay (ELISA) had a 98.2% sensitivity and a 91.7% specificity relative to the NDV HI test. The ELISA values regressed significantly (F = 930, df = 1/362, P less than 0.001) on the hemagglutination-inhibition (HI) titers. The correlational coefficient was 0.85. For individuals, two tests can have the same result based upon chance alone. Kappa is a measure of agreement between two tests that corrects for this chance agreement. The kappa between the ELISA and HI test was calculated to be 0.84 (Z = 7.74, P = 0.00001), which indicates a highly significant agreement between the two tests.     

Monoclonal-antibody-mediated enzyme-linked immunosorbent assay for detection of reticuloendotheliosis viruses

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of reticuloendotheliosis viruses (REVs). The assay uses a mixture of monoclonal antibodies (MCAs) prepared against a 62-kilodalton REV envelope glycoprotein (gp62) to capture antigen, rabbit anti-REV serum as detection antibody, and peroxidase-conjugated anti-rabbit IgG as indicator antibody. The MCAs were reactive with REV strain T, chick syncytial virus, and duck infectious anemia virus but unreactive against Marek's disease and avian leukosis viruses. The ELISA was compared with complement fixation test and REV immunofluorescent assay of infected fibroblasts, plasmas, and egg albumen from infected chickens. The lower limit of gp62 detection was about 120 ng of REV protein. Limit dilution of infectious REV was detected after 7-8 days of cultivation of infected fibroblasts.     

间接酶联免疫吸附试验(ELISA)检测血清2型鸭疫里默氏杆菌抗体的研究

鸭疫里默氏杆菌(Riemerella anatipestifer，RA)感染是危害养鸭业的最重要传染病之一，该病主要呈急性败血症或慢性过程，以纤维素性心包炎、肝周炎、气囊炎和干酪性输卵管炎为特征。RA的血清型很多，其中血清2型在我国的发生频率很高，危害非常严重。目前已建立的用于检测RA抗体的方法有琼脂扩散试验、凝集试验、ELISA等，这些检测方法或是灵敏度不高，或是由于使用抗原成分的不同而存在一些不足，在我国仅仅建立了用于血清1型RA抗体的检测。为能有效对血清2型RA抗体进行血清流行病学调查以及其疫苗的免疫效果进行监测和评价，开展本项研究。     

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.     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody.

To detect antibody against feline herpesvirus 1 (FHV-1) in the sera of cats, the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using nuclear antigen was investigated. The standardized optical density readings (ODs) of the ELISA obtained by the 1-step serum dilution (1:80) method were compared with the serum neutralization test (SNT) results, with a correlation of 0.993, and with the hemagglutination inhibition (HI) test results, with a correlation of 0.851. The ODs for the ELISA titers were obtained using the serial serum dilution method and were compared with the SNT results, with a correlation of 0.933, and with the HI test results, with a correlation of 0.987. In the experimental infection of 4 specific-pathogen-free cats, the results of different serologic tests (SNT and HI) and the ELISA using the serial serum dilution method revealed rapid production of antibodies after inoculation, whereas the ELISA using the one-step serum dilution method indicated that titers increased more slowly. These results indicate that with the present ELISA using nuclear antigen, there are fewer demands on time and labor, making the method convenient for monitoring FHV-1 infection.