Differential diagnosis of PRV-infected versus vaccinated pigs using a novel EuNPs-virus antigen probe-based blocking fluorescent lateral flow immunoassay.

A novel time-resolved fluorescence blocking lateral flow immunoassay (TRF-BLFIA) was developed for on-site differential diagnosis of pseudorabies virus (PRV)-infected and vaccinated pigs using europium nanoparticles (EuNPs)-labeled virion antigens and high titer PRV gE monoclonal antibodies (PRV gE-mAb). Upon application of a positive serum sample, the specific epitopes of gE protein on the EuNPs-PRV probe were blocked, inhibiting binding to the PRV gE-mAb on the T line, resulting in low or negligible fluorescence signal, whereas when a negative sample was applied, EuNPs-PRV probes would be able to bind the antibody at the T line, leading to high fluorescence signal. Under optimized conditions, TRF-BLFIA provided excellent sensitivity and selectivity. When testing swine clinical samples (n = 356), there was 96.1% agreement between this method and a most widely used commercial gE-ELISA kit. Moreover, our method was rapid (15 min), cost-efficient and easy to operate with simple training, allowing for on-site detection. Thus, TRF-BLFIA could be a practical tool to differentially diagnose PRV-infected and vaccinated pigs.

Immune-tolerizing procedure for preparation of monoclonal antibodies against glycoprotein E of Pseudorabies virus.

The glycoprotein E (gE) of pseudorabies virus (PRV) is known to be an important marker protein in the control and eradication of Aujeszky's disease. In this study, BALB/c mice were immunized with gE-deleted PRV as tolerogen and with wild-type PRV as immunogen. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. Two hybridoma cell lines that could stably secrete the monoclonal antibody (MAb) against gE were achieved by using indirect ELISA screening and subcloning three times; they were named 1D2 and 2B2. Indirect immunofluorescence assay (IFA) revealed that the MAbs were specifically against gE of PRV. MAbs 1D2 and 2B2 were subgroup IgG1. The MAbs obtained in this study provide useful tools for the development of differential diagnostic methods for PRV.

Development of a convenient immunochromatographic strip for the diagnosis of infection with Japanese encephalitis virus in swine.

Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV). It is a major public health problem in Asia. JEV infects swine which results in fatal encephalitis, abortion and stillbirth in pregnant sow, and hypospermia in boars. Swine is a viral amplifier, and thus plays a critical role in JEV transmission. Thus, development of a rapid method for JEV detection in swine is required for clinical JE diagnosis, as well as to suppress viral spread. In this study, a convenient and rapid immunochromatographic strip (ICS) was developed for detecting JEV in swine using two monoclonal antibodies (MAbs) (2A2 and 4D1) against the E protein of JEV. Results showed that colloidal gold-conjugated MAbs 2A2 (CG-MAb) bond with JEV and the resulting complex was held by the other MAb 4D1 at the test line to give a reddish-purple band. Sensitivity tests demonstrated that ICS can detect 2.5x10(5)PFU of JEV. The clinical screening results showed that the specificity and sensitivity of the ICS were 99.3% and 85.7% respectively as compared to that of RT-PCR. This suggests that the MAbs-based ICS test can be used as a convenient method for the rapid detection of JEV in infected swine samples.

An indirect sandwich ELISA for the detection of avian influenza H5 subtype viruses using anti-hemagglutinin protein monoclonal antibody.

A sandwich ELISA test using AIV H5 subtype specific monoclonal antibody (clone 2H4) to an epitope of hemagglutinin protein has been developed. The monoclonal antibody was used to capture the antigen from clinical samples (swabs and tissues). Captured antigens from clinical samples were detected using polyclonal sera, purified AIV H5N1 particles were titrated in the sandwich ELISA and the limit of detection was determined to be approximately 1.0 ng of influenza viral protein in virus preparations. Fifteen AIV strains of H1-H15 subtypes and some other pathogens were tested by this system, and the test is specific to H5 subtype viruses as it failed to detect other AIV subtype viruses and other pathogens. Varieties of clinical samples originating from laboratory experiments (n=382) and from fields (n=288) were employed to test the efficacy of DAS-ELISA test. The test compared very well with the traditional method for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs. In comparison to virus isolation the sensitivity and specificity of sandwich ELISA were found to be 98.6% and 97.6% respectively. In addition, the DAS-ELISA was used to test samples of experimentally infected birds and clinical samples obtained from central China in 2005. The assay proved to be sensitive and specific for the rapid detection of AIV H5 subtype virus form the tissues and swabs in infected animals.

Avian influenza (H5N1) virus in waterfowl and chickens, central China.

In 2004, 3 and 4 strains of avian influenza virus (subtype H5N1) were isolated from waterfowl and chickens, respectively, in central People's Republic of China. Viral replication and pathogenicity were evaluated in chickens, quails, pigeons, and mice. We analyzed the sequences of the hemagglutinin and neuraminidase genes of the isolates and found broad diversity among them.

A latex agglutination test for the rapid detection of avian influenza virus subtype H5N1 and its clinical application.

A rapid and simple latex agglutination test (LAT) for the detection of avian influenza virus (AIV) subtype H5N1 in chicken allantoic fluids, tracheal swabs, and tissues was developed. Monoclonal antibodies against the hemagglutinin glycoprotein of H5N1 were covalently coupled onto the surface of carboxylated latex bead using a water-soluble carbodiimide to obtain sensitized latex particles (SLP). These SLPs strongly agglutinated in the presence of allantoic fluid containing H5N1, but not fluids containing other AIV sub-types such as HIN1, H3N2, H4N6, and H9N2. Using this LAT, the virus was detectable in tracheal swabs 24 hours to 30 days after inoculating chickens with H5N1, with detection rates ranging from 45.5 to 79.2%. Much higher rates of detection were obtained from tissues collected postmortem from H5N1 experimentally infected chickens; lung tissue yielded the highest detection rate (96.7%), followed by kidney, spleen, brain, and liver tissues (90%). Lower detection rates were achieved with heart (41.7%) and cloacal tissues (26.8%). When the LAT was compared with other detection methods, the agreement with the viral isolation, H5 antigen immunochromatographic test,and H5 real-time RT-PCR test was 93.97, 95.18, and 87.95%, respectively. The test was highly specific for H5N1 in chickens and water fowls and had sensitivity comparable to other diagnostic tests evaluated.

Different neutralization efficiency of neutralizing monoclonal antibodies against avian influenza H5N1 virus to virus strains from different hosts.

BALB/c mice were immunized with formalin-treated influenza A/CK/Hubei/327/2004 virus. Six monoclonal antibodies specific to HA were selected, designed 1H8, 1D11, 2B7, 2C9, 2H4 and 4C9, respectively. The six Mabs probed linear epitopes by western blot assays. In ELISA additivity assays, the low additivity indexes (< or =28.3) of each pair Mabs indicated that the epitopes recognized by the six Mabs were located on the globular head of HA1. The neutralization activity of anti-HA1 Mabs and chicken polyclonal sera to various AIV H5N1 strains from different hosts was followed by virus neutralization with MDCK cells. All Mabs except 2C9 and chicken polyclonal serum showed highest neutralizing activity to lowly virulent A/Duck/XF/XFY/2004 from different phylogenetic lineage, and lowest neutralization efficiency to highly virulent A/CK/XF/XFJ/2004. For the other two highly virulent viruses, 1D11, 2H4, 4C9 and chicken polyclonal sera had higher neutralization to A/Goose/ZF/ZFE/2004 than A/CK/Hubei/327/2004, and 1H8 and 2B7 had considerable level of neutralizing efficiency to them. These findings suggested that the neutralizing antibodies showed lower neutralization efficiency to highly virulent virus strains than lowly virulent virus strains and strong cross-neutralizing reaction between virus strains located in different phylogenetic lineages. Moreover, the neutralizing Mabs could more efficiently neutralize AIV H5N1 strains from the natural hosts generally, such as waterfowl.

[Preparation and partial characterization of monoclonal antibodies specific for nuclear protein of avian influenza virus].

AIM: To prepare the monoclonal antibodies (mAbs) specific for nuclear protein (NP) of avian influenza virus (AIV) and identify their biological properties. METHODS: BALB/c mice were immunized with AIV (formaldehyde-inactivated AIV H9N2, Triton X-100-lysed H9N2 and AIV NP expressed in E.coli, respectively). Hybridoma cell lines secreting anti-AIV NP mAbs were developed through cell fusion, screening and cloning. The mAb's titer was determined by indirect ELISA. Specificity of mAbs was identified by cross-reaction test and indirect immuno-fluorescence assay (IFA). RESULTS: 6 hybridoma cell lines secreting anti-AIV NP mAbs were obtained, designated 4F4, 1C3, 1G11, 1C2, 1D10 and 2F7. ELISA detection showed that the titers of two mAbs (1G11 and 1D10) out of 6 mAbs were the highest (2(-13) and 2(-14), respectively) and their specificity was also better than that of the others, confirmed by cross-reaction test and IFA. CONCLUSION: In this study 6 mAbs against AIV NP were obtained. The mAbs 1G11 and 1D10 perform the best in titer and specificity. This work paves the way for AIV study and development of method for rapid detection of AIV.

Generation of monoclonal antibodies and epitope mapping of ApxIVA of Actinobacillus pleuropneumoniae.

To study functions of ApxIV, a species-specific and in vivo inducible RTX toxin identified in Actinobacillus pleuropneumoniae recently, and to develop a diagnostic trial distinguishing the pigs infected naturally and vaccinated with inactivated and/or subunit vaccines, we attempted to prepare monoclonal antibodies against ApxIV. BALB/c mice were immunized with ApxIVAN and ApxIVAC which are N- and C-terminal halvies (814 and 997 amino acids, respectively) of ApxIVA produced in E. coli BL21 (DE3), respectively. Eight monoclonal antibodies were selected, four (designated as 1A8, 1G5, 3E7 and 4H9) against ApxIVAN and another four (named as 1B12, 2E5, 4D8 and 4G2) against ApxIVAC. Western blot and ELISA additivity assays suggested that all monoclonal antibodies except 1A8 are specific to the corresponding immunogen, 1A8 reacts with both immunogens which have a overlapping region of 156 residues. ELISA additivity tests revealed that at least five epitopes in ApxIV are defined by eight monoclonal antibodies, two between 1 and 866 amino acids, one between 867 and 1022 amino acids and two between 1023 and 1863 amino acids. In conclusion, we have succeeded in producing eight monoclonal antibodies, which react with five different epitopes of ApxIV.