[Development of a flow cytometry method for detection of bovine multi-cytokines].

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.

Enhanced therapeutic efficacy of Listeria-based cancer vaccine with codon-optimized HPV16 E7.

Cervical cancer is a leading cause of high mortality in women in developing countries and has a serious impact on women's health. Human papilloma virus (HPV) prophylactic vaccines have been produced and may hold promise for reducing the incidence of cervical cancer. However, the limitations of current HPV vaccine strategies make the development of HPV therapeutic vaccines particularly important for the treatment of HPV related lesions. Our previous work has demonstrated that LM4Δhly::E7 was safe and effective in inducing antitumor effect by antigen-specific cellular immune responses and direct killing of tumor cell on a cervical cancer model. In this study, the codon usage effect of a novel Listeria-based cervical cancer vaccine LM4Δhly::E7-1, was evaluated for effects of codon-optimized E7 expression, cellular immune response and therapeutic efficacy in a tumor-bearing murine model. Our data demonstrated that up-regulated expression of E7 was strikingly elevated by codon usage optimization, and thus induced significantly higher Th1-biased immunity, lymphocyte proliferation, and strong specific CTL activity ex-vivo compared with LM4Δhly::E7-treated mice. Furthermore, LM4Δhly::E7-1 enhanced a remarkable therapeutic effect in establishing tumors. Taken together, our results suggest that codon usage optimization is an important consideration in constructing live bacterial-vectored vaccines and is required for promoting effective T cell responses.

WbaP is required for swarm motility and intramacrophage multiplication of Salmonella Enteritidis spiC mutant by glucose use ability.

Salmonella spp. can survive and replicate in macrophage cells to cause persistent infection, SpiC is a necessary T3SS effector, but its pathogenic mechanism is still not known completely. In our study, Salmonella Enteritidis spiC mutant (SEΔspiC) was found to have stronger swarming motility and intramacrophage hyperproliferation which was closely related to glucose metabolism. SEΔspiC wbaP::Tn5 mutant was screened out by transposon mutagenesis, which had weaker swarming motility and intramacrophage replication ability than SEΔspiC in the presence of glucose. Bioinformatics displayed that undecaprenyl-phosphate galactose phosphotransferase (Wbap), encoded by wbaP gene, was a key enzyme for glucose metabolism and Lipopolysaccharide(LPS) synthesis, which confirmed our outcome that Wbap was involved in intramacrophage replication ability by glucose use in addition to swarming motility based on SEΔspiC. This discovery will further promote the understanding of the interaction between wbaP gene and spiC gene and the intracellular Salmonella replication mechanism.

inlF Enhances Listeria monocytogenes Early-Stage Infection by Inhibiting the Inflammatory Response.

The internalin family proteins, which carry the leucine repeat region structural motif, play diverse roles in Listeria monocytogenes (Lm) infection and pathogenesis. Although Internalin F, encoded by inlF, was identified more than 20 years ago, its role in the Lm anti-inflammatory response remains unknown. Lm serotype 4b isolates are associated with the majority of listeriosis outbreaks, but the function of InlF in these strains is not fully understood. In this study, we aimed to elucidate the role of inlF in modulating the inflammatory response and pathogenesis of the 4b strain Lm NTSN. Strikingly, although inlF was highly expressed at the transcriptional level during infection of five non-phagocytic cell types, it was not involved in adherence or invasion. Conversely, inlF did contributed to Lm adhesion and invasion of macrophages, and dramatically suppressed the expression of pro-inflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF-α). Consistent with the in vitro results, during Lm infection mice, inlF significantly inhibited the expression of IL-1ß and IL-6 in the spleen, as well as IL-1ß, IL-6, and TNF-α in the liver. More importantly, inlF contributed to Lm colonization in the spleen, liver, and ileum during the early stage of mouse infection via intragastric administration, inducing severe inflammatory injury and histopathologic changes in the late stage. To our knowledge, this is the first report to demonstrate that inlF mediates the inhibition of the pro-inflammatory response and contributes to the colonization and survival of Lm during the early stage of infection in mice. Our research partly explains the high pathogenicity of serovar 4b strains and will lead to new insights into the pathogenesis and immune evasion of Lm.

The PA-interacting host protein nucleolin acts as an antiviral factor during highly pathogenic H5N1 avian influenza virus infection.

Polymerase acidic (PA) protein is a multifunctional regulator of influenza A virus (IAV) replication and pathogenesis. In a previous study, we reported that nucleolin (NCL) is a novel PA-interacting host protein. In this study, we further explored the role of NCL during highly pathogenic H5N1 avian influenza virus infection. We found that depletion of endogenous NCL in mammalian cells by siRNA targeting during H5N1 infection resulted in significantly increased viral polymerase activity, elevated viral mRNA, cRNA and vRNA synthesis, accelerated viral replication, and enhanced apoptosis and necrosis. Moreover, siRNA silencing of NCL significantly exacerbated the inflammatory response, resulting in increased secretion of IL-6, TNF-α, TNF-ß, CCL-4, CCL-8, IFN-α, IFN-ß and IFN-γ. Conversely, overexpression of NCL significantly decreased IAV replication. Collectively, these data show that NCL acts as a novel potential antiviral factor during H5N1 infection. Further studies exploring the antiviral mechanisms of NCL may accelerate the development of new anti-influenza drugs.

A Promising Listeria-Vectored Vaccine Induces Th1-Type Immune Responses and Confers Protection Against Tuberculosis.

Deaths associated with tuberculosis (TB) is rising and accounted for 1.4 million deaths in 2015 many of which were due to drug-resistant bacteria. Vaccines represent an important medical intervention, but the current Bacilli Calmette-Guerin (BCG) vaccine is not ideal for the protection of teenagers and adults. Therefore, a safe and effective vaccine is urgently needed. In this study, we designed a novel vaccine using an attenuated Listeria monocytogenes strain carrying fusion antigen FbpB-ESAT-6 (rLM) and characterized its safety and protective efficacy against Mycobacterium tuberculosis (M.tb) infection in mice. Compared to the wild type strain yzuLM4 and parental strain LMΔactA/plcB (LM1-2), the virulence of rLM was significantly reduced as judged by its infectious kinetics and LD50 dose. Further characterization of intravenous immunization showed that prime-boost vaccination significantly increased the levels of Th1 cytokines (IFN-γ, IL-17, and IL-6), and enhanced cytotoxic T lymphocyte (CTL) CTLs activity, suggesting that rLM could elicit potent Th1/Th17 responses. More importantly, rLM significantly conferred the protection against M.tb H37Rv challenge. Collectively, our findings indicated that rLM is a novel and useful tool to prevent M.tb infection, and can be potentially be used to boost BCG-primed immunity.

[Expression and assembly of chimeric flagellins in Escherichia coli BL21(DE3) and Salmonella].

Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1ß secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.

A Genetically Modified attenuated Listeria Vaccine Expressing HPV16 E7 Kill Tumor Cells in Direct and Antigen-Specific Manner.

Attenuated Listeria monocytogenes (L. monocytogenes, LM) induces specific CD8+ and CD4+ T cell responses, and has been identified as a promising cancer vaccine vector. Cervical cancer is the third most common cancer in women worldwide, with human papillomavirus (HPV), particularly type 16, being the main etiological factor. The therapeutic HPV vaccines are urgently needed. The E7 protein of HPV is necessary for maintaining malignancy in tumor cells. Here, a genetically modified attenuated LM expressing HPV16 E7 protein was constructed. Intraperitoneal vaccination of LM4Δhly::E7 significantly reduced tumor size and even resulted in complete regression of established tumors in a murine model of cervical cancer. We provided evidence that recombinant LM strains could enter the tumor tissue and induce non-specific tumor cell death, probably via activation of reactive oxygen species and increased intracellular Ca2+ levels. LM4Δhly::E7 effectively triggered a strong antigen-specific cellular immunity in tumor-bearing mice, and elicited significant infiltration of T cells in the intratumoral milieu. In summary, these data showed LM4Δhly::E7 to be effective in a cervical cancer model and LM4Δhly::E7 induced an antitumor effect by antigen-specific cellular immune responses and direct killing of tumor cells, indicating a potential application against cervical cancer.

[Construction and characterization of an attenuated recombinant Listeria monocytogenes vector vaccine delivering HPV16 E7].

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD50) of LM4△hly::E7 strain was 3.863×109 CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.

[Expression and immunogenicity of Ag85A protein of Mycobacterium tuberculosis].

Objective: The aim of this study was to express Mycobacterium tuberculosis Ag85A protein and to evaluate its immunogenicity in mice. Methods: The cold expressed system and chaperone competent cells BL21 were combined to express Mycobacterium tuberculosis Ag85A protein, then the protein was purified with affinity chromatography and identified by Western Blot analysis. Results: The immunogenicity of the purified Ag85A protein was evaluated in C57BL/6 mice. Results show that high level of specific IgG was elicited in the serum, and the splenocytes and lymph node cells of immunized mice could produce more Th1 cytokines, such as IFN-γ and TNF-α, after stimulated with specific antigen. Conclusions: Ag85A protein can induce strong specific humoral and Th1 type cellular immune responses, providing an important biological material for further research and application.

[Inflammasome responses in macrophages induced by C-terminal peptides of Escherichia coli EscI protein].

OBJECTIVE: To analyze NLRC4 inflammasome responses in macrophages induced by C-terminal of Escherichia coli EscI protein. METHODS: NLRC4 inflammasome responses in mouse peritoneal macrophages were analyzed after delivery of the peptides containing C-terminal amino acid sequences of E. coli EscI protein in vitro. RESULTS: The peptides containing C-terminal 15 amino acids of EscI protein could significantly activate NLRC4 inflammasome responses in macrophages pre-stimulated with lipopolysaccharide. Intracellular caspase-1 was activated and pyroptotic dead cells were found after peptides delivery. The contents of cytokines, IL-1ß and IL-18, in supernatants were elevated significantly compared with that of the control (P < 0.05). Besides, through comparison of IL-1ß contents under different stimulation conditions, 4 h incubation after peptides delivery (peptides: lipofectamine 2000 = 70 µg/µL) could obviously promote the secretion of IL-1ß. CONCLUSION: Peptides containing C-terminal 15 amino acids of E. coli EscI protein can significantly induce NLRC4 inflammasome activation in macrophages.

[Immunological characteristics of Mycobacterium tuberculosis antigen Rv2628].

Antigen Rv2628 of Mycobacterium tuberculosis is associated with latent tuberculosis infection. In this study, Rv2628 was prokaryotic expressed and purified, its immunological characteristics was evaluated with macrophage cell line RAW264.7 and BALB/c mice. The results show that Rv2628 was mainly expressed in form of inclusion body confirmed by SDS-PAGE, and could react with rabbit anti-H37Rv polyclonal antibody detected by Western blotting assay, indicating that the protein had an effective immunoreactivity. The interactions between Rv2628 and macrophage cell line RAW264.7 confirmed that it could effectively induce cells to produce pro-inflammatory cytokines, the relative expression level of IL-6 mRNA was higher than the control group in 1-12 h. BALB/c mice were subcutaneously immunized with Rv2628 protein, the production of IFN-gamma and IL-4 in the spleen cells was determined by Sandwich ELISA, in the Rv2628 immunized group, the level of IFN-gamma was significantly higher than that of IL-4 (P < 0.000 1). It indicated the protein induced Th1-tendency immune responses. At the same time, Rv2628(11-30) peptide used as coating antigen, the murine serum antibody titer detected by indirect-ELISA was 1:1 600, which demonstrated that Rv2628 could also induce humoral immune responses. In summary, Rv2628 could induce specific pro-inflammatory cytokines, affectively induce strongly Th1-tendency immune response and humoral response, it could be a potential target for developing subunit vaccine against TB. In addition, it laid foundation for probing the cross-talk between M. tb and host.

[Prokaryotic expression and identification of HPV16 E7 protein].

HPV16 E7 fusion protein was expressed in E. coli BL21, and its applied value for HPV was evaluated. HPV16 E7 gene was amplified by PCR, and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21, and HPV16 E7 fusion was expressed through IPTG induction. The expressed product was analyzed by SDS-PAGE and Western blot, subsequently purified according to Glutathione Sepharose 4B purification procedure. An indirect ELISA with the purified fusion protein as the coating antigen was then established to detect E7 serum antibodies from mice immunized with recombinant Listeria monocytogenes delivering HPV16 E7. The results demonstrated that the soluble fusion protein was highly expressed at 25 degrees C after induction with 0.5 mM IPTG. Furthermore, the result of Western blot analysis showed that the fusion protein had good specific reaction with an anti-E7 monoclonal antibody. Indirect ELISA result confirmed that the fusion protein could detect the serum antibodies against E7 with a titer of 1:200. The expressed GST-E7 fusion protein was immunocompetent, which was useful in the research of E7 biological function and therapeutic vaccine.

[Protective immune responses induced by a recombinant Listeria monocytogenes delivering HPV16 E7].

OBJECTIVE: To investigate specific immune responses elicited by a recombinant Listeria monocytogenes strain LM4 deltahly::E7 and assess protective effect in C57BL/6 mice. METHODS: C57BL/6 mice were intraperitoneally immunized with LM4 deltahly:: E7 at 1-week intervals. After the second immunization, cellular immunity elicited by this recombinant L. monocytogenes strain was analyzed via an ELISPOT assay, Cytotoxic T lymphocytes (CTL) measurement assay and analysis of effector T cells proportion in the splenocytes. Also, the serum antibodies against HPV16 E7 protein were determined in an ELISA assay. Finally, protective effect was assessed against the challenge with TC-1 tumor cells. RESULTS: The immune responses elicited by LM4 deltahly::E7 were biased towards Th1 type in the ELISPOT assay. Also, LM4 deltahly::E7 was able to induce E7-specific CTL activity, with average specific lysis of 72%, which was highly significant difference compared with the controls (P<0.01). Moreover, the proportion of effector T cells in the spleens were increased in mice immunized with recombinant L. monocytogenes strain (P<0.05). The titer of E7-specific antibodies in mice immunized with LM4 delta hly::E7 was 1:400 in the ELISA assay. Furthermore, immunization with LM deltahly::E7 protected all mice against the lethal tumor cell challenge. CONCLUSION: The data suggest that attenuated Listeria monocytogenes delivering HPV16 E7 antigen could induce both E7-specific cell mediated immunity and humoral immunity, and had a protective effect against challenge with TC-1 tumor cells.

[Construction and characterization of an ompR gene deletion mutant from Salmonella enteritidis].

OBJECTIVE: To investigate the role of ompR gene from Salmonella enteritidis in biofilm formation and virulence. METHODS: We constructed an ompR mutant of Salmonella enteritidis by suicide plasmid pGMB151. Biofilm forming ability of the mutant was detected by crystal violet assay and scanning electron micrography. Virulence of the mutant was determined by assay of adherence to and invasion of epithelial cells, and mouse challenge experiments. RESULTS: The ompR mutant was confirmed by RT-PCR and the pattern of outer membrane protein. The mutant did not produce cellulose, curli, and biofilm, and showed similar adherence percentage to and invasion percentage of epithelial cells as wild type strain. In addition, intraperitoneal challenge of bacteria in BALB/c mice revealed that LD50 of the mutant strain was 10(6.67) CFU, while that of the wild type strain was less than 2 CFU. CONCLUSION: These data indicate that the ompR gene is involved in both biofilm formation and virulence in Salmonella enteritidis.

[Construction of recombinant adenovirus expressing Ag85B of Mycobacterium bovis and its cellular immunoproperties in mice].

OBJECTIVE: We constructed recombinant adenovirus rAd-Ag85B expressing antigen 85B of Mycobacterium bovis and analyzed cellular immune responses after mice immunization. METHODS: We amplified the fbpB gene from genomic DNA of BCG by PCR and cloned into pMD18-T vector for sequence analysis. Then we constructed pDC516-Ag85B and co-transfected with plasmid pBHGfrtdeltaE1, 3FLP into Ad293 cells by lipofectamine 2000. Then we identified the recombinant Ad-Ag85B virus by RT-PCR, Western blot assay and TEM observation after negative staining. BALB/c mice were subcutaneously immunized with the dose of 10(8) TCID50 rAd-Ag85B or rAd. Two weeks after immunization, CD69 expression, T lymphocyte proliferation and ELISPOT were detected in spleen cells of immunized mice. RESULTS: We obtained the correct recombinant adenovirus rAd-Ag85B expressed Ag85B antigen. The level of both splenic CD69+ CD4+ T and CD69+ CD8+ T cells of rAd-Ag85B group were significantly higher than those of rAd control. The SI index of rAd-Ag85B was much higher than rAd. In ELISPOT assay more IFN-gamma specific secreting cell spots other than IL-4 were detected in rAd-Ag85B group. CONCLUSION: The recombinant adenovirus rAd-Ag85B can induce specific Th1 cell immune responses in mice.

A reusable capacitive immunosensor for detection of Salmonella spp. based on grafted ethylene diamine and self-assembled gold nanoparticle monolayers.

Fabrication of a novel capacitive immunosensor based on grafted ethylene diamine and self-assembled gold nanoparticle monolayer on glassy carbon electrode for the detection of Salmonella spp. is described for the first time. In the present study, the Salmonella spp. monoclonal antibodies (denoted as McAbs) was immobilized on gold nanoparticles. Interaction of McAbs and Salmonella spp. was detected directly using the electrochemical impedance spectroscopy (EIS) technique. The experimental results showed that the concentration of antigen was measured through the relative change in capacitance in the corresponding specific binding of Salmonella spp. and McAbs. Under the optimized conditions, the relative changes in capacitance were proportional to the logarithmic values of Salmonella spp. concentrations in the range of 1.0 x 10(2) to 1.0 x 10(5) CFU mL(-1) (r = 0.991) with the detection limit of 1.0 x 10(2) CFU mL(-1). The stability of proposed immunosensor could be estimated by determining the relative change in capacitance, which remained almost the same in two months and decreased gradually to 85.3% of initial value after four months' storage. The used immunosensor could be regenerated repeatedly by immersing in glycine-HCl buffer solution (pH 2.8). Finally, the proposed immunosensor was successfully used for the detection of Salmonella spp. in lab-processed commercial pork samples.

[The construction of reporter plasmid of ULBP1 and preliminary studying on the influence of NS3/4A on transcription of ULBP1].

AIM: To construct a report vector of ULBP1 promoter gene and preliminary study on the influence of NS3/4A on transcription of ULBP1. METHODS: ULBP1 core promoter sequence was amplified by PCR, and inserted into pGL3-enhance vector, constructing ULBP1 reporter plasmid pGL3-ULBP1; The HCV protease NS3/4A gene was subcloned into pcDNA3-Flag vector (pcDNA3-Flag-NS3/4A), and the expression of this plasmid was demonstrated by Western blot. The influence of inhibition by NS3/4A on the level of ULBP1 transcription was tested by assaying the Luciferase activity in cells transfected with pGL3-ULBP1 with Luminometer. RESULTS: The reporter plasmid of ULBP1 promoter gene and the eukaryotic expression plasmid Flag-NS3/4A have been constructed and expressed successfully; and the protease NS3/4A inhibit the level of ULPB transcription. CONCLUSION: The protease NS3/4A of HCV down-regulates ULBP1 expression by inhibiting the transcription of ULBP1.

[Localization analysis of attenuated Salmonella typhimurium with oral immunization].

To analyze the localization of attenuated Salmonella typhimurium after oral immunization. Prokaryotic expression plasmid pYA33-DsRed, carrying the RFP gene, was constructed and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria were named as X4550 (33-DsRed). The macrophage cell line RAW264.7 and bone marrow dendritic cell (BMDC) were invaded by X4550 (33-DsRed) in vitro. Furthermore, BALB/c mice were immunized with recombinant bacteria orally. RFP positive cells (RFP+ cells) were detected by Flow Cytometry (FCM) from spleen, liver, Mesenteric lymp node (MLN), Peyer's patch (PP), Inguinal lymph node (ILN). The invasion rate increased when the multiplicity of infection(MOI) were improved in this two kinds of cells respectively. After oral immunization with X4550 (33-DsRed), RFP+ cells were detected by FCM on 1d, 2d, 3d, 5d, 7d in spleen, liver, MLN, PP, ILN cells. The first day, RFP+ cells were detected in MLN and PP, and in PP at a higher rate of 1.4% than that of MLN. 0.4% RFP+ cells were detected the next day in ILN. On 3th day, the rates of RFP+ cells were increased in all of above tissues or organs and decreased on the 5th day. At the 7th day, RFP+ cells couldn't be detected in all tissues or organs tested. It is suggested that the invasion ability and the transfer through mucosal pathway and targeting to recognize immune tissue or organs are favor of the research in mucosal vaccine and the vaccine efficiency.

[Development and characterization of monoclonal antibodies against H7 hemagglutinin of avian influenza virus].

AIM: To prepare monoclonal antibodies (mAbs) against the hemagglutinin protein of H7 subtype of avian influenza virus (AIV). METHODS: (6-8 weeks old) BALB/c mice of were immunized endermicly with H7 subtype of AIV. The splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells after the last immunization. Hybridoma cells were screened by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The reactivity and specificity of mAbs were evaluated by HI test and Western blot assay. RESULTS: Four hybridoma cell lines secreting specific mAbs named 2E2, 2A4, 5F5 and 7G5 were developed. The HI titers of these mAbs were 5 x 2(7)-5 x 2(11), and the immunoglobulin subclass of 2E2 was IgM, that of 2A4 was IgG1, and that of 5F5 and 7G5 was IgG2a. Western blot analysis confirmed that the mAbs only reacted with M(r) 75 000 HA protein of H7 subtype of AIV but did not react with the proteins of Newcastle disease virus (NDV). The results of HI reactivity assay suggested that 2E2, 5F5 and 7G5 only reacted with H7 subtype of AIV but did not react with other subtypes of AIV, NDV and infectious bronchitis virus (IBV). However 2A4 reacted not only with H7 subtype of AIV but also with H15N8 reference strain of AIV at low HI level. CONCLUSION: These mAbs can be used as a useful tool to analyze the HA structure of AIV. They also provide the effective reagents for the rapid detection of H7 subtype of AIV.