Diagnostic Efficacy of a Recombinant Cysteine Protease of Spirometra erinacei Larvae for Serodiagnosis of Sparganosis

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient''s sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.     

Identification of antigenic differences that discriminate between cattle vaccinated with Anaplasma centrale and cattle naturally infected with Anaplasma marginale

Monoclonal antibodies were raised against the vaccine strain of Anaplasma centrale used in Australia. A monoclonal antibody that reacted with an 80 kDa antigen was used to develop an A. centrale-specific fluorescent antibody test that will be useful for confirming species identity in patent infections. Another monoclonal antibody that reacted with a 116 kDa antigen was used to develop an A. centrale-specific competitive inhibition enzyme-linked immunosorbent assay (ELISA) for the serological identification of vaccinated cattle. The sensitivity of the ELISA was 100% in cattle experimentally infected with A. centrale, 97.1% in a vaccinated beef herd and 98.3% in a vaccinated dairy herd. The specificity of the ELISA was 98.6% in non-vaccinated cattle outside the Anaplasma marginale-endemic area, 97.9% in non-vaccinated cattle within the A. marginale-endemic area and 100% in cattle experimentally infected with A. marginale. The ELISA detected antibodies to A. centrale in cattle up to 9 years after vaccination with no apparent decrease in sensitivity. The assay has proved extremely valuable in Australia for investigating reported failures of multivalent live vaccines used to protect cattle against anaplasmosis and babesiosis, and should be similarly useful elsewhere in the world where these types of vaccines are used, e.g. Israel and South America.     

特异性卵黄抗体(IgY)对小鼠大肠杆菌败血症的保护作用

目的:研究特异性IgY对小鼠大肠杆菌败血症的保护作用.方法:以灭活的E.coli O111免疫产蛋母鸡,抗体经水稀释及盐析分离纯化.ELISA法检测大肠杆菌特异性IgY对大肠杆菌及LPS的结合活性.腹腔注射E.coli O111(1011cfu/mL)建立小鼠败血症模型,攻毒剂量为0.1mL/10g体重.小鼠随机分为5组,分别给药保护:空白组(生理盐水)、阴性对照组(非特异性IgY,20mg/mL)、阳性对照组(头孢哌酮20mg/mL)、高剂量组(特异性IgY,40mg/mL),低剂量组(特异性IgY,20mg/mL).给药剂量为:攻毒前,0.15mL/10g体重,每天一次,共两天;攻毒后,0.25mL/10g体重,每天一次,共七天.观察小鼠临床表现、体重变化、白细胞(WBC)和血小板(PLT)数变化及各组小鼠的死亡率.结果:特异性IgY与E.coli O111和LPS均有体外结合活性.大肠杆菌攻毒后,小鼠体重下降,各组小鼠外周血中WBC和PLT数均有不同程度的下降.特异性IgY保护组各项指标较快恢复到正常水平,其他组恢复缓慢.各组小鼠七天内的死亡率分别为:空白组与阴性对照组都为100%;阳性对照组60%;低剂量IgY组30%;高剂量IgY组10%.结论:特异性IgY对小鼠大肠杆菌败血症有保护作用.     

Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice

Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines. In this study, we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen (HA) from the strains A/New Caledonia/20/99 (H1N1) (pV1A5) and A/California/04/2009 (H1N1) (pVEH1), respectively. After verifying antigen expression, the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model. Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies. HA-specific T-cells were also measured in IFN-γ ELISpot assays. The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD(50) of mouse-adapted A/New Caledonia/20/99 (H1N1). Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses (P<0.05) than the pVAX1 control group. Additionally, the pV1A5 vaccine effectively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100% compared with a 40% survival rate in the pVEH1 vaccinated group (P<0.05). Our study indicates that the seasonal influenza DNA vaccine completely protects against the homologous A/New Caledonia/20/99 virus (H1N1), while the pandemic influenza DNA vaccine only partially protects against this virus.     

Quantitative enzyme-linked immunosorbent assay determination of an abundant hemoglobin-derived anti-infective peptide in human placenta

A fragment of the human β-chain of hemoglobin (HEM), hHEMβ111-146, was shown to have broad antimicrobial properties. The 3.9-kDa peptide was postulated to occur in high concentrations in placenta tissue. We established a reliable method to quantify hHEMβ111-146 in placenta tissue. Our methodology consists of a tissue extraction step (step 1), a chromatographic enrichment step (step 2), and a final quantification step (step 3) by enzyme-linked immunosorbent assay (ELISA). The specificity of the ELISA reaction was confirmed by parallel analysis of the samples via Western blot (step 4). The ELISA measured the absorbance of a tetramethylbenzidine substrate at 450 nm. It showed no cross-reactivity with the corresponding γ- and α-HEM regions and low cross-reactivity with the β-HEM region and full-length HEM. The sample preparation procedure enabled a prepurification of hHEMβ111-146, completely eliminating cross-reactive proteins and HEM peptides. The linear range of detection in step 3 was 20-200 ng/well (200-2000 μg/L) with a limit of quantification of 23 ng/well (230 μg/L) and a limit of detection of 7 ng/well (70 μg/L). The assay was characterized by good linearity (r² > 0.99), intraday precision (coefficient of variation [CV] = 2.2-8.3%), interday precision (CV = 1.8-9.1%), and accuracy (76-109%). The mean recovery of the ELISA was determined to be 97%, and the overall recovery during steps 1-3 was found to be 40.3 ± 2.5%. We measured concentrations from 0.28 to 0.74 mg/g placenta tissue of the hHEMβ111-146 in different placenta samples with an average concentration of 0.57 mg/g. This abundant concentration supports an important physiological role of hHEMβ111-146 in the placenta infective barrier.     

Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens

The efficacy of an oral DNA vaccine carrying the Eimeria tenella 5401 antigen gene delivered by attenuated Salmonella typhimurium was examined in an experimental challenge study. The DNA vaccine preparation was made by transforming the recombinant plasmid pcDNA3-5401 into the attenuated S. typhimurium strain (Dam(-) and PhoP(-)) (designated hereafter as ZJ111/pcDNA3-5401). The chickens were randomly divided into six groups, 50 per group. Group A were given PBS as control. Chickens in group B were fed with 10(8) colony forming units (CFU) of attenuated S. typhimurium carrying pcDNA3. Group C were immunised with 100 microg of the recombinant 5401 protein via intramuscular injection. Groups D to F orally received ZJ111/pcDNA3-5401 at doses of 10(7), 10(8) and 10(9)CFU per chicken, respectively. All immunisations were boosted 2 weeks later. The immunised chickens were challenged with 6x10(4) homologous sporulated oocysts 14 days after the second immunisation. No significant differences in body weight were detected between the groups before immunisation and at week 4 after the booster immunisation. The ZJ111/pcDNA3-5401 was eventually eliminated from the spleen and liver on week 6 post-immunisation. The plasmid pcDNA3-5401 was stably maintained in over 80% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunisation of chickens with ZJ111/pcDNA3-5401 elicited specific humoral responses and stimulated proliferation of peripheral blood lymphocytes. The lymphocyte proliferation response was significantly higher in all vaccinated groups than in the control chickens. Antibody response was significantly lower in group C than in groups immunised with strain ZJ111/pcDNA3-5401. Vaccination with the strain ZJ111/pcDNA3-5401 at 10(8) (group E) and 10(9) (group F) CFU per chicken provided 55.0 and 57.5% protection against E. tenella challenge, respectively. These results have important implications for the development of DNA vaccines against avian coccidiosis by bacteria-vectored oral delivery system.     

肿瘤患者血清中SARS-CoV抗体阳性原因分析

探讨SARS冠状病毒(SARS—CoV)抗体在SARS病原学诊断中的特异性及其在肿瘤患血清中的假阳性问题。应用ELISA和荧光定量RT-PCR技术检测了111例正常对照和40例肿瘤患血清中SARS—CoV抗体的阳性率。在111例正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3．6％(4／111);IgG抗体诊断SARS的特异性为96．4％,两种抗体同时阳性诊断SARS的特异性为100％。40例肿瘤患中,IgM抗体均阴性,IgG抗体阳性率17．5％(7／40)。经RT—PCR检测,上述肿瘤患阳性病例均为阴性。结果表明,同时测定SARS—CoV的两种抗体可降低诊断的假阳性率,提高诊断的特异性。用非纯化SARS—CoV抗原制备的ELISA试剂盒测定肿瘤患的SARS—CoV抗体,可能出现假阳性。在肿瘤患中出现假阳性的原因可能与包被的抗原有关。     

Comparison of Three ELISA Kits for the Differentiation of Foot-and-mouth Disease Virus-infected from Vaccinated Animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals.Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits,Ceditest(R)FMDV-NS (Ceditest(R) kit),UBI(R) FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI(R) kit) and a FMDV 3ABC-I-ELISA kitdeveloped at the Lanzhou Veterinary Research Institute.The test parameters (sensitivity and specificity) of the three kits were determined,and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits.The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest(R) kits was 98.05%,and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI(R) kits was 94.4%; the sensitivity of both Ceditest(R) and FMDV 3ABC-I-ELISA kit was 100%.However,the sensitivity of the UBI(R) kit was only 81.8%.With sera from naive or vaccinated non-infected animals,the specificity of all tests exceeded 90%.     

Comparison of three ELISA kits for the differentiation of foot-and-mouth disease virus-infected from vaccinated animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals. Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits, Ceditest® FMDV-NS (Ceditest® kit), UBI® FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI® kit) and a FMDV 3ABC-I-ELISA kit developed at the Lanzhou Veterinary Research Institute. The test parameters (sensitivity and specificity) of the three kits were determined, and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits. The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest® kits was 98.05%, and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI® kits was 94.4%; the sensitivity of both Ceditest® and FMDV 3ABC-I-ELISA kit was 100%. However, the sensitivity of the UBI® kit was only 81.8%. With sera from naive or vaccinated non-infected animals, the specificity of all tests exceeded 90%.     

Comparative evaluation of antibody positive titer by ELISA and IFA in Theileria annulata vaccinated cattle in Iran

An enzyme linked immunosorbent assay (ELISA) was used to evaluate antibody positive titer in vaccinated and non-vaccinated cattle using schizont infected myeloid cells as an antigen. The result was compared with indirect fluorescent antibody level in the same animals. For this study 116 milking cows, 95 vaccinated and 21 non-vaccinated, were bleeded in order to prepare sera. They were tested with both ELISA and IFA tests. 94 sera had positive antibody titer and 22 sera were negative through ELISA test but, with IFA test, only 89 sera showed positive antibody titer and 27 were negative. Thereby, it was concluded that the sensitivity and specificity of ELISA test in comparison with IFA test was 95.5% and 66.6% respectively. This study generally indicated that ELISA could be an effective test for sero-epidemiological investigations of bovine tropical theileriosis, and it is considered to be valid as an additional test to distinguish the vaccinated from the non vaccinated cattle in order to schedule vaccination programs.     

Determining Vaccination Frequency in Farmed Rainbow Trout Using Vibrio anguillarum O1 Specific Serum Antibody Measurements

Background

Despite vaccination with a commercial vaccine with a documented protective effect against Vibrio anguillarum O1 disease outbreaks caused by this bacterium have been registered among rainbow trout at Danish fish farms. The present study examined specific serum antibody levels as a valid marker for assessing vaccination status in a fish population. For this purpose a highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate sera from farmed rainbow trout vaccinated against V. anguillarum O1.

Study Design

Immune sera from rainbow trout immunised with an experimental vaccine based on inactivated V. anguillarum O1 bacterin in Freund’s incomplete adjuvant were used for ELISA optimisation. Subsequently, sera from farmed rainbow trout vaccinated with a commercial vaccine against V. anguillarum were analysed with the ELISA. The measured serum antibody levels were compared with the vaccine status of the fish (vaccinated/unvaccinated) as evaluated through visual examination.

Results

Repeated immunisation with the experimental vaccine lead to increasing levels of specific serum antibodies in the vaccinated rainbow trout. The farmed rainbow trout responded with high antibody levels to a single injection with the commercial vaccine. However, the diversity in responses was more pronounced in the farmed fish. Primary visual examinations for vaccine status in rainbow trout from the commercial farm revealed a large pool of unvaccinated specimens (vaccination failure rate = 20%) among the otherwise vaccinated fish. Through serum analyses using the ELISA in a blinded set-up it was possible to separate samples collected from the farmed rainbow trout into vaccinated and unvaccinated fish.

Conclusions

Much attention has been devoted to development of new and more effective vaccines. Here we present a case from a Danish rainbow trout farm indicating that attention should also be directed to the vaccination procedure in order to secure high vaccination frequencies necessary for optimal protection with a reported effective vaccine.     

NanoFerrite particle based radioimmunonanoparticles: binding affinity and in vivo pharmacokinetics

Dextran and PEG-coated iron oxide nanoparticles (NP), when suitably modified to enable conjugation with molecular targeting agents, provide opportunities to target cancer cells. Monoclonal antibodies, scFv, and peptides conjugated to 20 nm NP have been reported to target cancer for imaging and alternating magnetic field (AMF) therapy. The physical characteristics of NPs can affect their in vivo performance. Surface morphology, surface charge density, and particle size are considered important factors that determine pharmacokinetics, toxicity, and biodistribution. New NanoFerrite (NF) particles having improved specific AMF absorption rates and diameters of 30 and 100 nm were studied to evaluate the variation in their in vitro and in vivo characteristics in comparison to the previously studied 20 nm superparamagnetic iron oxide (SPIO) NP. SPIO NP 20 nm and NF NP 30 and 100 nm were conjugated to (111)In-DOTA-ChL6, a radioimmunoconjugate. Radioimmunoconjugates were conjugated to NPs using 25 microg of RIC/mg of NP by carbodiimide chemistry. The radioimmunonanoparticles (RINP) were purified and characterized by PAGE, cellulose acetate electrophoresis (CAE), live cell binding assays, and pharmacokinetics in athymic mice bearing human breast cancer (HBT 3477) xenografts. RINP (2.2 mg) were injected iv and whole body; blood and tissue data were collected at 4, 24, and 48 h. The preparations used for animal study were >90% monomeric by PAGE and CAE. The immunoreactivity of the RINP was 40-60% compared to (111)In-ChL6. Specific activities of the doses were 20-25 microCi/2.2 mg and 6-11 microg of mAb/2.2 mg of NP. Mean tumor uptakes (% ID/g +/- SD) of each SPIO 20 nm, NF 30 nm, and 100 nm RINP at 48 h were 9.00 +/- 0.8 (20 nm), 3.0 +/- 0.3 (30 nm), and 4.5 +/- 0.8 (100 nm), respectively; the ranges of tissue uptakes were liver (16-32 +/- 1-8), kidney (7.0-15 +/- 1), spleen (8-17 +/- 3-8), lymph nodes 5-6 +/- 1-2), and lung (2.0-4 +/- 0.1-2). In conclusion, this study demonstrated that 100 nm NF NP could be conjugated to (111)In-mAb so that the resulting RINP had characteristics suitable for AMF therapy. Although 100 nm RINP targeted tumor less than 20 nm SPIO RINP, their heating capacity is typically 6 times greater, suggesting the 100 nm NF RINP could still deliver better therapy with AMF.     

The effect of adjuvant and specific or non-specific vaccination on development of protective immunity of rabbits against Trichostrongylus colubriformis infection.

Adult New Zealand rabbits were vaccinated subcutaneously with one dose of 100 micrograms adult nematode phosphate buffered saline-soluble proteins (PBS-ASP, groups I and II), a detergent-soluble fraction of adult somatic proteins (DS-ASP, group III) or three doses of 1 mg normal rabbit serum proteins (group IV). Injections of the immunogens in groups II, III and IV were accompanied with beryllium hydroxide, Be(OH)2 as an adjuvant. Vaccinated rabbits and also those of group V (naive) were challenged orally with 10,000 infective larvae of T. colubriformis 14 days after antigen injection and necropsied 2 weeks later. A single dose of PBS-ASP induced 33.5% protection when the antigen was given alone (group I) and 69.4% when injected with Be(OH)2 (group II). A detergent-soluble fraction of ASP given with the adjuvant provided 87.2% protection (group III), whilst non-specific vaccination with serum proteins plus Be(OH)2 elicited 99% protection (group IV). Mesenteric lymph node leukocyte responses were measured using a leukocyte migration inhibition assay. A significant response was observed only in group IV. In ELISA tests IgA antibodies specific to PBS-ASP reached the highest level in the intestinal mucosa of groups I and II and in the bile of groups I and III. Antibody levels of IgG isotype were similar in the intestinal mucosa of all the immunized groups. Nematode antigen was detected using a 'sandwich' ELISA method in faecal protein extracts of rabbits of groups II and III on days 2-6 after challenge.     

网片重建治疗盆腔器官脱垂的临床疗效及安全性评价

目的:探讨经阴道放置网片的全盆底重建术治疗重度子宫脱垂的临床疗效和安全性。方法:选择2010年7月至2015年6月在青岛大学附属医院因重度盆腔器官脱垂接受全盆底重建术的患者126例,回顾性分析患者的临床资料、生活质量评分及并发症等相关资料。结果:126例患者平均手术时间(67.18±18.00)min,出血量(56.27±26.47)m L,留置尿管(3.47±0.94)天,住院天数(4.21±0.93)天。术后尿潴留的发生率为3.96%、阴道壁血肿0.79%、术后肢体疼痛7.14%、下肢静脉血栓0.79%,所有患者术中均无膀胱或直肠损伤等严重并发症的发生。126例患者中,111例完成随访(88.09%,111/126),随访时间为6~48个月,中位随访时间为27个月。术后发生网片暴露1例(0.9%,1/111),网片挛缩1例(0.9%,1/111),慢性盆腔疼痛2例(1.80%,2/111)、新发尿失禁6例(5.4%,6/111)。其中3例为急迫性尿失禁(2.7%,3/111),3例压力性尿失禁(2.7%,3/111)。盆底重建术后共有3例患者出现复发,其中2例因症状明显再次行手术治疗,客观治愈率的为97.29%(108/111),主观治愈率为98.19%(109/111)。与术前相比,术后6个月、12个月生活质量评分(PFDI-20)均较前均明显降低(P0.05)。术后21例患者恢复性生活,性生活疼痛者3例(14.28%),性生活不适者4例(19.04%),总体性生活满意度为85.71%(18/21)。结论:全盆重建术治疗重度盆腔器官脱垂的疗效较好,且网片相关并发症的发生率较低。     

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.     

Detailed study of anaerobic digestion of Spirulina maxima algal biomass

Biomass of the blue-green alga Spirulina maxima was converted to methane using continuous stirred tank digesters with an energy conversion efficiency of 59%. Digesters were operated using once-a-day feeding with a retention time (theta) between 5 and 40 days, volatile solid concentrations (S(to)) between 20 and 100 kg VS/m(3), and temperatures between 15 and 52 degrees C. The results indicated a maximum methane yield of 0.35 m(3) (STP)/kg VS added at theta 30 days and S(to) 20 kg VS/m(3). Under such conditions, the energy conversion of the algal biomass to methane was 59%. The maximum methane production rate of 0.80 m(3) (STP)/m(3) day was obtained with theta= 20 days and S = 100 kg VS/m(3). The mesophilic condition at 35 degrees C produced the maximum methane yield and production rate. The process was stable and characterized by a high production of volatile acids (up to 23, 200 mg/L), alkalinity (up to 20, 000 mg/L), and ammonia (up to 7000 mg/L), and the high protein content of the biomass produced a well buffered environment which reduced inhibitory effects. At higher loading rates, the inhibition of methanogenic bacteria was observed, but there was no clear-cut evidence that such a phenomenon was due to nonionized volatile acids or gaseous ammonia. The kinetic analysis using the model proposed by Chen and Hashimoto indicated that the minimum retention time was seven days. The optimum retention time increased gradually from 11 to 16 days with an increase in the initial volatile solid concentration. The kinetic constant K decreased with the improvement in the digester performance and increased in parallel with the ammonia concentration in the culture media.     

An ELISA based on the repeated foot-and-mouth disease virus 3B epitope peptide can distinguish infected and vaccinated cattle

To develop a strategy of differentiating infected from vaccinated animals (DIVA) with foot-and-mouth disease virus (FMDV), a short (27aa) peptide containing three conserved linear B cell epitopes of the FMDV 3B nonstructural protein was designed. This novel BF peptide was synthesized using a gene splicing by overlap extension protocol with preferred codons for Escherichia coli. The resultant eight tandem repeat multimer (1, 2, 4, 6, 8, 16, 24, and 32BF) were expressed as soluble fusion proteins in E. coli. An indirect ELISA was developed based on the recombinant 8BF protein with the aim of specifically distinguishing antibodies induced by FMDV infection but not those induced by vaccination. Using the cut-off value of 0.3, the sensitivity of the assay was 96.8% and the specificities for naive and vaccinated cattle were 99.8 and 99.0%, respectively. The performance of the newly developed epitope-based ELISA was compared with three commercial NSP ELISA kits. The 8BF-ELISA appears to be a promising DIVA test for FMD control and eradication.     

Extensive serum biomarker analysis in the prethrombotic state of recurrent spontaneous abortion

The prethrombotic state (PTS) is a possible cause of recurrent spontaneous abortion (RSA). The aim of this study was to identify serum biomarkers for the detection of RSA with PTS (PSRSA). A Quantibody array 440 was used to screen novel serum-based biomarkers for PSRSA/NRSA (RSA without PTS). Proteins differentially expressed in PSRSA were analysed using bioinformatics methods and subjected to a customized array and enzyme-linked immunosorbent assay (ELISA) validation. We used receiver operating characteristic to calculate diagnostic accuracy, and machine learning methods to establish a biomarker model for evaluation of the identified targets. 20 targets were selected for validation using a customized array, and seven targets via ELISA. The decision tree model showed that IL-24 was the first node and eotaxin-3 was the second node distinguishing the PSRSA and NRSA groups (an accuracy rate of 100% and an AUC of 1). Epidermal growth factor (EGF) as the node distinguished the PSRSA and NC groups (an accuracy rate of 100% and an AUC of 1). EGF as the node distinguished the NRSA and NC groups (an accuracy rate of 96.5% and an AUC of 0.998). Serum DNAM-1, BAFF, CNTF, LAG-3, IL-24, Eotaxin-3 and EGF represent a panel of promising diagnostic biomarkers to detect the PSRSA.