GENE ENGINEERING EB VIRUS MEMBRANE ANTIGEN IN DETECTION OF MA-IgA ANTIBODY(COMPARISON WITH VCA-IgA AND EA-IgA ANTIBODIES)

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluo-rescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pretreatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Preparation and Preliminary Identification of Fluorescein Labeled Monoclonal Antibody against Canine Distemper Virus

[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

Monitoring Report of Maternal Antibody of Broiler Avian Influenza Virus H5 Subtype Re-8 Strain

[ Objective ] The paper was to prevent the occurrence of broiler avian influenza virus HS subtype Re-8 strain effectively in the breeding process of broilers. [Method] The maternal antibodies of broilers in Beijing Baochen Hongwang farm were monitored. According to the disappearance law of maternal antibody, the optimal immune time of broiler avian influenza virus H5 subtype Re-8 strain was determined. [ Result] The maternal antibody level of 2-day-old broilers was relatively high, concentrated at 6 log2 -9 log2, and the antibody positive rate was 100%. The maternal antibody level of 8-day-old broilers concentrated at 4 log2 -6 log2, and the antibody positive rate was 100%. The maternal antibody level of 17-day-old broilers concentrated at 0 log2 -3 log2 , and the antibody positive rate was 0. The average maternal antibody level of 24 - 37 days old broilers was 〈 1 log2, and the antibody positive rate was 0. [ Conclusion ] Although the av- erage maternal antibody level of 8-day-old broilers was higher than 5 log2 , 20% of chickens was 4 log2, and maternal antibody could not protect the flock completely. Therefore, the best primary immunization day age of chicks against avian influenza virus was 8 - 10 days of age.

Plasmid DNA encoding neutralizing human monoclonal antibody without enhancing activity protects against dengue virus infection in mice

Objective: To evaluate the expression of DNA plasmid-harboring modified antibody gene that produces neutralizing human monoclonal antibodies against four serotypes of dengue virus(DENV) without enhancing activity in BALB/c mice. Methods: We constructed pFUSE-based vectors(pFUSE1 G7 C2hVH and pFUSE1 G7 C2hVL) containing genes encoding the variable domains of the heavy or light chain of the anti-dengue virus antibody 1 G7 C2, a human IgG1 that has been characterized for its neutralizing activity to DENV-1-4. Leucine(L) at positions 234 and 235 on the Fc CH2 domain in pFUSE1 G7 C2hVH was mutated to alanine(A)(LALA mutation) by site direct mutagenesis, and the new plasmid was termed pFUSE1 G7 C2hVHLALA. An equal amount of pFUSE1 G7 C2hVL and 1 G7 C2hG1-LALA plasmids were co-transfected into Chinese hamster ovary cells(CHO-K1) and a single dose of 100 μg 1 G7 C2hG1-LALA plasmid was intramuscularly injected, followed by electroporation in BALB/c mice. The secreted 1 G7 C2hG1-LALA antibodies in cell culture supernatant and mouse serum were examined for their biological functions, neutralization and enhancing activity. Results: The co-transfection of heavy-and light-chain 1 G7 C2hG1-LALA plasmids in CHO-K1 cells produced approximately 3 900 ng/mL human IgG and neutralized 90%-100% all four DENV, with no enhancing activity. Furthermore, the modified human IgG was produced more than 1 000 ng/mL in mouse serum on day 7 post plasmid injection and showed cross-neutralization to four DENV serotypes. Subsequently, antibody production and neutralization decreased rapidly. Nevertheless, the secreted neutralizing 1 G7 C2hG1-LALA in mouse serum demonstrated complete absence of enhancing activities to all DENV serotypes. Conclusions: These findings reveal that a new modified 1 G7 C2h G1-LALA expressing plasmid based on gene transfer is a possible therapeutic antibody candidate against DENV infection.

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Citrus yellow vein clearing virus （CYVCV） is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein （CP） of CYVCV Chongqing isolate （CYVCV- CQ） was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody （MAb） production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10-6 to 10-7 as determined by indirect enzyme-linked immunosorbent assay （ELISA）. Three serological assays, including dot enzyme-linked immunosorbent assay （dot-ELISA）, tissue blot-ELISA, and double-antibody sandwich （DAS）-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1：2 560 and 1：10 240 （w/v, g mL^-1）, respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not＇detected in any sample collected from Zhejiang or Jiangxi Province, China.

Preparation and Initial Application of a Monoclonal Antibody Specific for a Newly Discovered Conserved Linear Epitope of Rabies Virus Nucleoprotein

Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate （FITC） after purification and used in fluorescent antibody test （FAT）. Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line （BSR）. Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies

Safety of hepatitis B virus core antibody-positive grafts in liver transplantation: A single-center experience in China

BACKGROUND Given the shortage of suitable liver grafts for liver transplantation, proper use of hepatitis B core antibody-positive livers might be a possible way to enlarge the donor pool and to save patients with end-stage liver diseases. However, the safety of hepatitis B virus core antibody positive(HBcAb+) donors has been controversial. Initial studies were mainly conducted overseas with relatively small numbers of HBcAb+ liver recipients, and there are few relevant reports in the population of China's Mainland. We hypothesized that the safety of HBcAb+ liver grafts is not suboptimal.AIM To evaluate the safety of using hepatitis B virus(HBV) core antibody-positive donors for liver transplantation in Chinese patients.METHODS We conducted a retrospective study enrolling 1071 patients who underwent liver transplantation consecutively from 2005 to 2016 at West China Hospital Liver Transplantation Center. Given the imbalance in several baseline variables, propensity score matching was used, and the outcomes of all recipients were reviewed in this study.RESULTS In the whole population, 230 patients received HBcAb+ and 841 patients received HBcAb negative(HBcAb-) liver grafts. The 1-, 3-and 5-year survival rates in patients and grafts between the two groups were similar(patient survival: 85.8% vs 87.2%, 77.4% vs 81.1%, 72.4% vs 76.7%, log-rank test, P = 0.16; graft survival: 83.2% vs 83.6%, 73.8% vs 75.9%, 70.8% vs 74.4%, log-rank test, P = 0.19). After propensity score matching, 210 pairs of patients were generated. The corresponding 1-, 3-and 5-year patient and graft survival rates showed no significant differences. Further studies illustrated that the post-transplant major complication rates and liver function recovery after surgery were also similar. In addition, multivariate regression analysis in the original cohort and propensity score-matched Cox analysis demonstrated that receiving HBcA b+ liver grafts was not a significant risk factor for long-term survival. These findings were consistent in both HBV surface antigen-positive(HBsAg+) and HBsA g negative(HBsAg-) patients.Newly diagnosed HBV infection had a relatively higher incidence in HBsAg-patients with HBcAb+ liver grafts(13.23%), in which HBV naive recipients suffered most(31.82%), although this difference did not affect patient and graft survival(P = 0.50 and P = 0.49, respectively). Recipients with a high HBV surface antibody(anti-HBs) titer(more than 100 IU/L) before transplantation and antiviral prophylaxis with nucleos(t)ide antiviral agents post-operation, such as nucleos(t)ide antiviral agents, had lower de novo HBV infection risks. CONCLUSION HBcA b+ liver grafts do not affect the long-term outcome of the recipients. Combined with proper postoperative antiviral prophylaxis, utilization of HBcAb+ grafts is rational and feasible.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Maternal Antibody Protected Chicks from Growth Retardation and Immunosuppression Induced by Early Reticuloendotheliosis Virus Infection

To determine if the maternal antibody from breeders vaccinated with cell culture-adapted reticuloendotheliosis virus （REV） could protect chicks from early REV infection, one-day-old chicks with or without anti-REV maternal antibodies were inoculated with REV, and then their growth rates and antibody titers to Newcastle disease virus （NDV） and avian influenza virus （AIV）, after vaccination with inactivated vaccines, were compared. This study indicated that REV infection could cause growth retardation and severely inhibit immune reactions to inactivated vaccines against NDV and Avian influenza virus （AIV, H9 and H5） in one-day-old broilers without maternal antibodies specific to REV. Maternal antibody from breeders vaccinated with an attenuated REV vaccine effectively protected REV-challenged birds from growth retardation and immunosuppression on antibody reactions to NDV and AIV vaccines. Four weeks after vaccination, the HI titers to NDV, AIV-H9, and AIV-H5 in maternal antibody positive and negative groups were 3.36 ＋- 2.04 versus 1.58± 1.69 （P〈0.01）, 6.27±3.87 versus 0.71 ± 1.60 （P〈0.01）, and 6.72±3.92 versus 0.54± 1.44 （P〈0.01）. Maternal antibodies from breeders vaccinated with REV vaccine could successfully protect chicks from REV infection and effectively prevent REV-induced growth retardation and immunosuppression in antibody responses to NDV and AIV.

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

Mortality rates from a Nigerian isolate of the <i>Infectious Bursa Disease Virus</i>and passive haemagglutination antibody titer that protects chicks against challenge with the virus isolate

To determine passive haemagglutination (PHA) antibody titer that would protect chicks against Nigerian isolates of the Infectious Bursa Disease Virus (IBDV), five groups of chicks aged 30 days which had different antibody titers were challenged with a Nigerian isolate of virulent IBDV. Mortality rates of the different groups were plotted against their respective mean PHA antibody titers. A group with zero antibody titer had a mortality rate of 75% while those with PHA antibody titers of 185.6, 243.2, 256 and 307.2 had mortality rates of 40%, zero, zero and zero respectively. Linear equation generated for a line of best fit of the graph of mortality rates of the chicks on their IBD antibody titers gave antibody titer (X) at which mortality (Y) would be zero as 300. A mortality of 75% and the high antibody level needed to protect chicks suggest that the isolate may be a hypervirulent strain.

Monoclonal antibody-based serological detection of potato virus M in potato plants and tubers

Potato virus M(PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide. To investigate and control this viral disease, efficient and specific detection techniques are needed. In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies(MAbs). Four highly specific and sensitive murine MAbs, i.e., 1 E1, 2 A5, 8 A1 and 17 G8 were prepared through a conventional hybridoma technology. Using these four MAbs, we have developed an antigen-coated plate(ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers. PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240(w/v, g mL^(–1)) by the dot-ELISA or at 1:163 840(w/v, g mL^(–1)) by the ACP-ELISA. The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection. Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China. The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing. We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM. These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.

Rabies Virus Neutralizing Activity,Safety,and Immunogenicity of Recombinant Human Rabies Antibody Compared with Human Rabies Immunoglobulin in Healthy Adults

Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus （WSSV） in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based mieroarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the mieroarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62μg/mL, with a woven long shelf life for 6 months at 4℃ or 8 months at -20℃. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.

Application of GP5 Protein to Develop Monoclonal Antibody against Porcine Reproductive and Respiratory Syndrome Virus

In this study, a panel of monoclonal antibodies （mAbs） against Porcine reproductive and respiratory syndrome virus（PRRSV）, named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV （TCID50=5.5）, screened by the indirect ELISA and subjected to several limiting dilutions, mAbs were then identified by biological characterization. Among the two fusion cell strains, 8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass. The titers in cell culture supematant and abdomen liquor reached to 1：104and 1：105, respectively. The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively, and no reaction with Classical swine fever virus （CSFV） or Swine vesicular disease virus （SVDV）. The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa, respectively. In neutralization activity tests, the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1：512, but mAB 8C9 has no neutralization activities to PRRSV.

Adenovirus-expressed preS2 antibody inhibits hepatitis B virus infection and hepatic carcinogenesis

AIM:To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV in-fection and HBV-associated hepatic carcinogenesis.METHODS:An adenoviral vector carrying the full-length light and heavy chains of the HBV preS2Ab gene,Ad315-preS2Ab,was constructed.Enzyme linked immunosorbent assay (ELISA) and Western blotting analyses were used to determine the preS2Ab expres-sion levels in vitro.Immunofluorescent techniques were used to examine the binding affinity between the expressed HBV preS2Ab and HBV-positive liver cells.ELISAs were also used to determine hepatitis B surface antigen (HBsAg) levels to assess the inhibitory effect of the preS2Ab against HBV infection in L02 cells.The inhibitory effect of preS2Ab against hepatic carcinogen-esis was studied with diethylnitrosamine (DEN)-induced hepatocellular carcinomas (HCCs) in HBV transgenic mice.RESULTS:The expression of HBV preS2Ab increased with increases in the multiplicity of infection (MOI) of Ad315-preS2Ab in L02 cells,with 350.87 ± 17.37 μg/L of preS2Ab when the MOI was 100 plaque forming units (pfu)/cell.The expressed preS2Abs could recog-nize liver cells from HBV transgenic mice.ELISA results showed that L02 cells expressing preS2Ab produced less HBsAg after treatment with the serum of HBV pa-tients than parental L02 cells expressing no preS2Ab.HBV transgenic mice treated with Ad315-preS2Ab had fewer and smaller cancerous nodes after induction with DEN than mice treated with a blank Ad315 vec-tor or untreated mice.Additionally,the administration of Ad315-preS2Ab could alleviate hepatic cirrhosis and decrease the serum levels of alanine transaminase and aspartate transaminase.CONCLUSION:Adenovirus-mediated HBV preS2Ab expression could inhibit HBV infection in L02 cells,and then inhibit DEN-induced hepatocellular carcinogenesis and protect hepatic function in HBV transgenic mice.