A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs

Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no “gold standard” assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.     

Differential diagnosis of the infection caused by wild-type or CD2v-deleted ASFV strains by quantum dots-based immunochromatographic assay

African swine fever (ASF), a highly contagious and lethal disease, poses a tremendous threat and burden to the swine industry worldwide. Lack of available vaccines or treatments leaves rapid diagnosis as the key tool to control the disease. Quantum dots (QDs) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In this study, we developed a quantum dots-based fluorescent immunochromatographic assay (QDs-FICA) using CD2v as the diagnosis antigen to detect ASFV antibodies. The titre of the test strip was 1 : 5·12 × 10⁵. In addition, the strip was highly specific to anti-ASFV serum and had no cross-reaction with CSFV, PPV, PRRSV, PCV-2, PRV and FMDV. Moreover, a comparative test of 71 clinical samples showed that the coincidence rate was 85·92% between the test strip and the commercial ELISA kit (coated with p30, p62 and p72). The QDs-FICA can be used to detect ASFV antibodies, which is meaningful for the surveillance, control and purification of ASF.     

非洲猪瘟病毒p35蛋白作为诊断抗原的抗原性比较

为了筛选出酶联免疫吸附测定(Enzyme linked immunosorbent assay,ELISA)反应性最佳的非洲猪瘟病毒(African swine fever virus,ASFV)诊断抗原,通过建立ELISA方法,以杆状病毒昆虫细胞表达系统表达的ASFV p30蛋白诊断抗原为参照,首次探讨原核表达系统表...     

猪源支气管败血波氏杆菌百日咳杆菌黏附素基因的原核表达及其抗体检测ELISA方法

[目的]以猪源支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)百日咳杆菌黏附素(PRN)基因的原核表达产物为抗原建立检测PRN抗体的间接ELISA方法.[方法和结果]利用谷胱甘肽-S-转移酶(GST)表达系统对PRN基因在大肠杆菌中进行融合表达.SDS-PAGE和Western blot检测证实该基因获得高效表达,产物易于纯化且具有良好的免疫学活性.通过凝血酶酶切GST-PRN并回收,获得不含GST载体蛋白的PRN蛋白片段.以PRN蛋白片段为抗原建立检测天然PRN抗体的间接ELISA方法.该方法对猪巴氏杆菌病等7种常见细菌性疾病阳性血清的检测结果均为阴性,其敏感性比乳胶凝集试验提高4～128倍,能检测到人工感染14 d后的仔猪血清抗体IgG,对临床送检的1,229份猪血清的检测阳性率为32.7%.ELISA方法对阳性猪场的监测结果预示了保育期仔猪的合群导致猪群大量感染支气管败血波氏杆菌.[结论]该方法具有特异性强、敏感性高、重复性好的特点,可用于猪群PRN抗体水平监测和猪波氏菌病流行病学调查.     

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.     

Porcine Immunoglobulin Fc Fused P30/P54 Protein of African Swine Fever Virus Displaying on Surface of S. cerevisiae Elicit Strong Antibody Production in Swine

African swine fever virus(ASFV) infects domestic pigs and European wild boars with strong, hemorrhagic and high mortality. The primary cellular targets of ASFV is the porcine macrophages. Up to now, no commercial vaccine or effective treatment available to control the disease. In this study, three recombinant Saccharomyces cerevisiae(S. cerevisiae) strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S. cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated. To be specific, the P30-Fcc and P54-Fca fusion proteins displaying on surface of S. cerevisiae cells were produced by fusing the Fc fragment of porcine immunoglobulin Ig G1 or IgA1 with p30 or p54 gene of ASFV respectively. The recombinant P30-Fcc and P54-Fca fusion proteins expressed by S. cerevisiae were verified by Western blotting, flow cytometry and immunofluorescence assay.Porcine immunoglobulin Fc fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine. The absorption and phagocytosis of recombinant S. cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage(PAM) cells were significantly enhanced, too. Here, we introduce a kind of cheap and safe oral S. cerevisiae-vectored vaccine, which could activate the specific mucosal immunity for controlling ASFV infection.     

Development and Comparative Evaluation of a Competitive ELISA with Rose Bengal Test and a Commercial Indirect ELISA for Serological Diagnosis of Brucellosis

The development of a competitive ELISA for the detection of brucella-specific antibodies in bovines is described. Anti-brucella guinea pig serum was used as a source of competing antibodies. Lipo-polysaccharide purified from inactivated B. abortus S19 culture was used as antigen for the development of the assay. Sera from cattle were used in the competitive ELISA, rose bengal test and a commercial indirect ELISA. The following cattle sera were tested: (i) known positive sera (n = 80) (ii) known negative sera (n = 100) and (iii) field sera (n = 1184). Based on the receiver operating characteristics curve analysis and frequency distribution of the percentage of inhibition, 30% inhibition was considered the cut-off for positive and negative results. The sensitivity and specificity estimate on comparison with the commercial indirect ELISA was 94.87 and 92.12% respectively. The competitive ELISA described is a simple method for the routine screening of animal sera for detecting Brucella-specific antibodies.     

A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs

African swine fever(ASF) is a devastating infectious disease in swine that is severely threatening the global pig industry. An efficacious vaccine is urgently required. Here, we used the Chinese ASFV HLJ/18 as a backbone and generated a series of genedeleted viruses. The virulence, immunogenicity, safety, and protective efficacy evaluation in specific-pathogen-free pigs,commercial pigs, and pregnant sows indicated that one virus, namely HLJ/18-7GD, which has seven genes deleted, is fully attenuated in pigs, cannot convert to the virulent strain, and provides complete protection of pigs against lethal ASFV challenge.Our study shows that HLJ/-18-7GD is a safe and effective vaccine against ASFV, and as such is expected to play an important role in controlling the spread of ASFV.     

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei,a Tier 1 select agent

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.     

Development of luciferase-linked antibody capture assay based on luciferase immunoprecipitation systems for antibody detection of porcine reproductive and respiratory syndrome virus

Background

Early detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies.

Results

Known anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result.

Conclusion

The LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.

     

CTB与HCV抗原决定簇融合蛋白的抗原性及其在抗HCV ELISA试剂盒中的应用

系统研究了通过霍乱毒素B亚基与HCV的4个抗原决定簇进行基因融合所表达的12种融合蛋白中HCV抗原决定簇的反应原性,探索了以融合蛋白为抗原,进行抗-HCV检测的途径。结果表明,多数融合蛋白中HCV抗原决定簇均能与对应的HCV抗体结合。以融合蛋白95082为抗原研制的抗-HCV ELISA试剂检测122名献血员血清,结果与美国雅培公司抗-HCV ELISA试剂检测的结果完全一致,经药品生物制品检定所检定,其特异性、灵敏度、精密性及稳定性均达到国家卫生部抗-HCV ELISA试剂的暂行检定标准。     

Human serum antibody response to Helicobacter pylori whole cell antigen in an institutionalized Bangladeshi population

AIMS: To use a commercial ELISA kit and an immunoblot assay to investigate the antibody levels of selected members of the Bangladeshi population to Helicobacter pylori protein antigens. METHODS AND RESULTS: Using immunoblotting, high seroprevalence rates were observed in all age groups, although the subjects within the 1-9 years age group had the highest seroprevalence of antibodies to H. pylori antigens. By ELISA, the highest level of seroprevalence was observed in those over the age of 20 years. CONCLUSION: On the basis of these results the overall prevalence rate of H. pylori infection for the whole population was 77.4%; 77.9% for orphan boys and 76% for carers. CagA antibodies were detected in 86% of those with high levels of antibodies to H. pylori antigens. SIGNIFICANCE AND IMPACT OF THE STUDY: A combination of immunoblotting and ELISA was the most efficient means of detecting serum antibodies to H. pylori antigens and could be applied to the screening of human sera for H. pylori-specific antibodies.     

Recombinant cystatin-like protein-based competition ELISA for Trichinella spiralis antibody test in multihost sera

ObjectivesTrichinella spiralis is a zoonotic parasite with a complex parasitic life cycle and exposed to animals or humans by infectious meat. To control transmissions of T. spiralis through the food chain to humans, sensitive and selective multihost sera-diagnosis is urgent needed for monitoring T. spiralis exposure.MethodsA competition enzyme-linked immunosorbent assay (cELISA) for T. spiralis infection diagnosis in multihost sera was developed based on recombinant cystatin-like protein (rCLP-cELISA) as well as monoclonal antibodies. The sensitivity and accuracy of the rCLP-cELISA were quantified using swine (n = 1316), mice (n = 189) and human (n = 157) serum samples. T. spiralis-antibody targeting test ability of the rCLP-cELISA in swine (n = 22) and human (n = 36), instead of other parasites or viruses antibodies, was evaluated.ResultsThe rCLP-cELISA showed high agreement with commercial ELISA kits in field swine sera assessed by Cohen’s kappa value (κ = 0.7963). And it showed 100% specificity in human trichinellosis detection with sensitivity of 96.49%, no cross-reaction with other parasite or virus infections, and high positive detection rate of 87.5% in low-dose infected swine. Besides, the rCLP-cELISA exhibited potential in the detection of T. spiralis, T. nelsoni and Trichinella T8 infections.ConclusionsThe rCLP-cELISA can be used for T. spiralis-associated antibody test in multihost sera.     

Application of protein-A indirect ELISA (PAI-ELISA) for the detection of anti-Smith antibodies in systemic lupus erythematosus patients

An indirect form of protein-A ELISA (PAI-ELISA) was optimized and, when used to detect anti-Smith antibodies in sera of 31 systemic lupus erythematosus (SLE) patients, gave results comparable with those using a commercial immunodiffusion kit. The number of sera found to be positive for anti-Smith antibodies by ELISA was seven, four of which were also found positive by immunodiffusion.B.O. Siti-Rohana is with the Universiti Kebangsaan Malaysia (UKM), Faculty of Medicine, Kuala Lumpur, Malaysia. I.B. Ahmad is with the Department of Microbiology, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, Bangi, 43600 UKM, Malaysia; B.A. Nasaruddin is with the Institute for Medical Research, Malaysia.     

Development of a Disperse Dye Immunoassay Technique for Detection of Antibodies against Neospora caninum in Cattle

In this study a disperse dye immunoassay method was standardized and evaluated for detection of antibodies against Neospora caninum in cattle. Sera from 150 cattle with a recent history of abortion were collected and tested by commercial ELISA kit and a standardized in-house dye immunoassay system. The positivity rate for the sera used in this study was 34.6% for the disperse dye immunoassay (DDIA) compared to 32% obtained by ELISA kit. This study showed no significant difference between DDIA and ELISA. The results indicated that the DDIA provide an economic, simple, rapid and robust test for detection of N. caninum infection in cattle.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

非洲猪瘟病毒CP204L基因的原核表达与单抗制备

由非洲猪瘟病毒(ASFV)引起的非洲猪瘟(ASF)给我国养猪业带来了不可估量的经济损失,严重阻碍了我国养猪业的发展,研发ASFV快速诊断试剂是目前最重要的内容之一。CP204L基因编码ASFV结构蛋白p30。本研究以克隆ASFV的CP204L基因为基础,通过基因重组技术,加入His标签,将构建的重组质粒命名为pET-28a-CP204L。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,37℃经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达6h,表达蛋白进行SDS-PAGE鉴定和Western Blot检测。重组蛋白纯化后免疫小鼠制备筛选单克隆抗体,Western Blot和IFA验证单抗的结合特异性。结果表明,重组的pET-28a-CP204L诱导后表达蛋白为30kD,以不可溶性包涵体形式存在;表达蛋白利用His标签进行纯化,获得纯化蛋白2mg,单克隆抗体筛选获得5株IgG亚型的ASFV p30蛋白的单抗,且均具有良好的结合活性。本研究为发展ASFV检测方法提供了基础。     

Effect of O. porcinus Tick Salivary Gland Extract on the African Swine Fever Virus Infection in Domestic Pig

African swine fever is a haemorrhagic disease in pig production that can have disastrous financial consequences for farming. No vaccines are currently available and animal slaughtering or area zoning to restrict risk-related movements are the only effective measures to prevent the spread of the disease. Ornithodoros soft ticks are known to transmit the African swine fever virus (ASFV) to pigs in farms, following the natural epidemiologic cycle of the virus. Tick saliva has been shown to modulate the host physiological and immunological responses during feeding on skin, thus affecting viral infection. To better understand the interaction between soft tick, ASFV and pig at the bite location and the possible influence of tick saliva on pig infection by ASFV, salivary gland extract (SGE) of Ornithodoros porcinus, co-inoculated or not with ASFV, was used for intradermal auricular inoculation. Our results showed that, after the virus triggered the disease, pigs inoculated with virus and SGE presented greater hyperthermia than pigs inoculated with virus alone. The density of Langerhans cells was modulated at the tick bite or inoculation site, either through recruitment by ASFV or inhibition by SGE. Additionally, SGE and virus induced macrophage recruitment each. This effect was enhanced when they were co-inoculated. Finally, the co-inoculation of SGE and virus delayed the early local spread of virus to the first lymph node on the inoculation side. This study has shown that the effect of SGE was powerful enough to be quantified in pig both on the systemic and local immune response. We believe this model should be developed with infected tick and could improve knowledge of both tick vector competence and tick saliva immunomodulation.