Detection of schistosomiasis antibodies in urine patients as a promising diagnostic maker

Objective:To determine secreted antischistosoma antibodies in urine and to discern the epidemiological situation of schistosomiasis in the agricultural field labourers’camps city in the Gezira State-central Sudan.Methods:Total of 66 urine and 66 serum paired samples were collected from those who confirmed parasitologically positive and negative with schistosomiasis from the two camps.Samples were tested using ELBA technique to measure and compare the immunoglobulin G(IgG) levels in serum and urine samples of schistosomiasis patients.Results: The overall prevalence of S.mansoni and S.haematobium was 53.8%and 15.4%,while the intensity were(2.04 GMEC) and(0.9 GMEC) respectively.The relative percentage of positive IgG individulas in urine was 92.40%where as 96.97%in serum.Statistically no significant difference between the IgG levels in serum and urine samples was observed.Conclusions:This study shows that the detection of secreted IgG antibodies in urine can substitute serum for diagnosis of schistosomiasis.

An ELISA Based on a Truncated Soluble ORF2 Protein for the Detection of PCV2 Antibodies in Domestic Pigs

Postweaning multisystemic wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA) based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.

Combination of specific monoclonal antibodies allow identification of soluble aggregates of by sandwich ELISA

Aggregate amyloid beta protein1-42 (Aβ1-42) can typically be found in the early stage of Alzheimer’s disease (AD). Aβ1-42 self-assembles and is highly toxic to neurons. Thus, recognizing aggregated Aβ1-42 is very important for elucidation of Aβ1-42 structure and for the diagnosis of AD. In this study, the specificity of the 79-3 monoclonal antibody against soluble aggre- gate Aβ1-42 was measured by sandwich Enzyme-Linked Immuno Sorbent Assay (ELISA). Eight monoclonal antibodies against both soluble aggregates and amorphous aggregates were used as primary antibodies. Soluble aggregates and amorphous aggregates were used as antigen. As secondary antibody, HRP was labeled with the 79-3 monoclonal antibody. The reactivity of the 79-3 monoclonal antibody against soluble aggregates was confirmed in all combinations, but little reactivity against amorphous aggregates was found. Furthermore, we performed the above sandwich ELISA using the 37-11 antibody, which is reactive against large oval aggregates (LOA) that occur in micro aggregates, instead of the 79-3 antibody. The 77-3 antibody is 1 of the 8 monoclonal antibodies against soluble aggregates;amorphous aggregates also reacted with the 37-11 antibody. These results indicated that soluble aggregates are specifically recognized by a combination of different antibodies. The combined use of these antibodies can be applied to the diagnosis of AD and to defining the structure of the Aβ1-42.

A nanobody-based blocking enzyme-linked immunosorbent assay for detecting antibodies against pseudorabies virus glycoprotein E

Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.

牛传染性鼻气管炎病毒gB ELISA抗体与中和抗体相关性分析

牛传染性鼻气管炎病毒(IBRV)是牛传染性鼻气管炎(IBR)的病原体,常引起流产和呼吸道感染等综合征,给养牛业造成了巨大经济损失。gB作为IBRV中最保守的抗原蛋白,是IBRV诊断的重要靶蛋白。为探究牛gB–ELISA抗体水平与VNT效价的相关性,本研究采用阻断ELISA和VNT两种不同的方法对某一牛场50份血清进行IBRV gB ELISA抗体检测和中和抗体测定。gB ELISA结果显示,阳性血清19份,阴性血清31份;中和抗体结果阳性17份,阴性33份。通过统计学相关系数分析,gB抗体阻断率和中和抗体效价值的相关系数为1.0,从而说明IBRV gB ELISA抗体和中和抗体呈较强的正相关。本研究为使用IBRV gB ELISA抗体水平评估牛群中IBRV抗体保护效果提供理论依据。

FIELD STUDIES IN THE DIAGNOSIS OF SCHISTOSOMIASIS BY MONOCLONAL ANTIBODYDOT-ELISA

ＦＩＥＬＤＳＴＵＤＩＥＳＩＮＴＨＥＤＩＡＧＮＯＳＩＳＯＦＳＣＨＩＳＴＯＳＯＭＩＡＳＩＳＢＹＭＯＮＯＣＬＯＮＡＬＡＮＴＩＢＯＤＹＤＯＴ－ＥＬＩＳＡＬｏｎＷｅｄａｎ（娄文娴），ＺｈｕＸｕｍｉｎｇ（朱旭明），ＹａｎｇＳｈｉｊｉｎｇ（杨士静）．Ｄｅｐａｒｔｍ...

Development of Double Antibody Sandwich ELISA for Detection of Duck or Goose Flavivirus

In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1：3 200 and 1：160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1：10 000. The positive standard value was 0.247 （OD450.m）. The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL^-1. The ELISA had no cross-reaction with Newcastle disease virus （NDV）, Avian influenza virus （AIV）, Infectious bronchitis virus （IBV）, Infectious bursal disease virus （IBDV）, Duck hepatitis virus （DHV）, and Gosling plague virus （GPV）. Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.

Novel P22-monoclonal antibody based blocking ELISA for the detection of African swine fever virus antibodies in serum

African swine fever(ASF)is a highly infectious,transboundary viral disease of domestic and wild pigs,and is currently the most serious threat to world swine production,resulting in significant economic loss.In the absence of vaccines and treatments,the control of the disease entirely depends on accurate and early diagnosis accompanied by the culling of infected pigs.Thus,a highly specific and sensitive diagnostic assay is required during an outbreak and surveillance of the disease.In this study,a highly sensitive,specific,rapid and repeatable P22-monoclonal antibody-based blocking enzyme-linked immunosorbent assay(bELISA)assay was developed for the detection of antibodies against genotype I and II African swine fever viruses(ASFVs).A total of 806 pig serum samples were tested to evaluate the performance of the diagnostic assay.To determine the PI(percent Inhibition)cut-off value,receiver-operating characteristic(ROC)analysis was applied.According to the ROC analysis of the data,98.10%specificity and 100%sensitivity were recorded when the threshold cut-off value of PI was established at 47%.In addition,the assay was able to detect ASFV antibodies as early as 9 days post-infection when serum samples from experimentally infected pigs were used.Taking all together,the results of the present study indicated that the P22-mAb based bELISA assay can be used for rapid and accurate detection of antibodies against ASFV,which could play a valuable role in the containment and prevention of ASFV as an alternative to other serological diagnostic methods.Also,this study will assist researchers to further investigate the immunogenic importance of P22 protein in ASFV infection.

Study of Immunoassay Methods for Recombinant Human Erythropoietin (rhEPO) Using Competitive ELISA

Two different immunoassay methods, competitive indirect enzyme-linked immuno-sorbent assay (CI-ELISA) and amplificative competitive indirect ELISA (ACI-ELISA) using biotin-avidin complex system were studied to detect rhEPO. The linear ranges were 50-20000 ng/mL and 10-50000 ng/mL for CI-ELISA and ACI-ELISA, respectively. The low detection limits of CI-ELISA and ACI-ELISA were 62.8 ng/mL and 8.5 ng/mL, respectively.

国产抗-HIV ELISA诊断试剂质量提高的血清学证实

目的 了解国产抗 HIVELISA诊断试剂质量提高情况 ,保证检测结果的准确性。方法 使用卫生部艾滋病预防与控制中心艾滋病参比实验室的试剂质量考核血清盘 ,对 3种国产抗 HIVELISA试剂质量考核 ,并对结果比较分析。结果 2 0 0 1年生产的抗 HIVELISA诊断试剂敏感性比 1998、1999年的试剂明显提高 ,阳性漏检率明显减少。结论 国产抗 HIVELISA诊断试剂质量在不断提高 。

血清载脂蛋白A5 ELISA法的建立及评价

目的建立定量测定人血清中载脂蛋白A5(apoA5)浓度的ELISA双抗体夹心法,评价该法的敏感性、特异性、批内批间变异系数(CV),并分析apoA5与血脂指标的相关性。方法方阵法确定apoA5单克隆抗体的最佳检测浓度,建立apoA5标准曲线,ELISA双抗体夹心法测定重组apoA5蛋白,确定最低检测限及批内、批间CV,并检测300例正常人apoA5浓度和血脂指标,应用百分位数法计算apoA5的参考值。结果制备的apoA5单克隆抗体的最佳检测浓度为1∶800,最低检测限为2.12μg/L;特异性检测试验表明:apoA5单克隆抗体只针对重组apoA5蛋白和血清中天然的apoA5,apoA1和BSA的A值均<0.2;批内CV<3.5%,批间CV<5.5%。血清apoA5的参考值范围为255.92～383.81μg/L。相关性分析表明:apoA5与TG呈负相关(r=-0.27,P<0.05),与TC存在负相关趋势,与HDL-C呈正相关(r=0.36,P<0.05)。结论本实验室建立的ELISA双抗体夹心法灵敏度和特异性均较好,为apoA5的临床检测提供了可行的方法。

Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

Recently, serum Golgi protein 73 （GP73） levels have been found to be elevated in patients with hepatocellu- lar carcinoma （HCC）, and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent as- say （ELISA）. Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody （mAb） designated as 6A2 using recom- binant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blot- ting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls （P = 0.0036）. Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls （P = 0.0172）.

HRP-SPA ELISA检测猪日本乙型脑炎病毒抗体的研究

将盐析法提纯的、用Hela细胞系增殖的猪日本乙型脑炎病毒JapaneseBencephalitis(JEV)抗原包被酶标板,以HRP-SPA作为第二抗体,建立了检测JEV抗体的ELISA试验方法。结果表明,抗原的最适包被浓度为1∶800,待检血清最适稀释度为1∶50,HRP-SPA最佳工作浓度为1∶1000,各步反应时间确定为5min。交叉试验、阻断试验、重复性试验、敏感性试验证明本实验特异性强,重复性好,敏感性高。包被的病毒抗原经甲醛固定15min即可进行正式试验。确定其测定标准为:S/N≥14.81为阳性,S/N<12.53为阴性,12.53≤S/N<14.81为可疑。

Development of new lateral-flow immunochromatographic strip using colloidal gold and mesoporous silica nanoparticles for rapid diagnosis of active schistosomiasis

Objective: To develop a new sandwich based lateral flow immunochromatographic strip for rapid detection of circulating Schistosoma mansoni antigen in serum and urine samples of patients with active schistosomiasis. Methods: This lateral flow immunochromatographic strip was prepared by using anti-Schistosoma mansoni soluble egg antigen monoclonal antibody conjugated gold nanoparticles (MAb-AuNPs) as antigen-detecting antibody, while crystalline material (MCM)-41-MAb bioconjugate was immobilized at the test line as antigen-capturing antibody. Both antigen capturing and detecting antibodies formed sandwich complexes with circulating Schistosoma mansoni antigen in the positive samples. Sandwich complexes immobilized at the test line gave distinct red color. The assay reliability was examined by using urine and serum samples of 60 Schistosoma mansoni infected patients, 20 patients infected with parasites other than Schistosoma, and 20 healthy individuals as negative controls. Results were compared with those obtained via sandwich enzyme linked immunosorbent assay (ELISA). Results: The detection limit of circulating Schistosoma mansoni antigen by lateral flow immunochromatographic strip was lower (3 ng/mL) than the detection limit by ELISA (30 ng/mL). The sensitivity and specificity of lateral flow immunochromatographic strip in urine samples were 98.3% and 97.5%, respectively compared to 93.5% and 90.0% by ELISA. In serum samples, they were 100.0% and 97.5%, respectively compared to 97.0% and 95.0% by ELISA. The strip test took approximately 10 min to complete. Conclusions: This new lateral flow immunochromatographic strip offers a sensitive, rapid, and field applicable technique for diagnosis of active schistosomiasis.

测定尿激酶含量夹心法 ELISA 的建立

采用两种识别不同抗原决定簇的抗尿激酶(UK)单克隆抗体建立了测定人 UK 含量的夹心法ELISA.本法灵敏度为0.15ng/ml,批内 CV4.3%,批间 CV8.7%,平均回收率98%.应用本法测定了部分正常细胞和肿瘤细胞株培养上清中的 UK 含量,肿瘤细胞培养上清 UK 量明显高于正常细胞.测定82名正常人血浆中 UK 含量,其结果为1.31±0.6ng/ml.

用Dot- ELISA对猪瘟抗体水平监测的报告

采用猪瘟Dot-ELISA诊断试剂盒对湖北省3个规模化猪场猪瘟抗体水平进行监测,以了解通过目前我国常用猪瘟免疫程序注射疫苗后猪群的免疫状况;同时分析了目前我国猪瘟流行特点,即由急性爆发型向温和型猪瘟发展趋势,指出只有通过正确免疫程序,加强疫苗管理,采用综合防治,才能控制和消灭猪瘟;通过实验操作和结果表明Dot-ELISA猪瘟诊断试剂盒具有微量、简便、快速、敏感等特性,是适用于基层现场检测猪瘟较好方法之一.

Epidemiology of schistosomiasis in Gezira area Central Sudan and analysis of cytokine profiles

Objective:To determine and compare anti-schistosoma IgC,interleukin-10(IL-10) and interferon-γ(IFN-γ)levels in the serum of patients and endemic controls and to investigate the epidemiological situation of Al-Hebaika village in the northern part of Gezira Agricultural Irrigation Scheme in 2005.Methods:During 2005 survey,serum were collected from 118 villagers.Sixty eight were parasitological positive(patients),and 50 were negative(endemic controls).Indirect ELISA was used to measure and compare the levels of immunoglobulin C(IgC) against Schistsoma mansoni(5.mansoni) soluble worm antigen(SWA) in the patients and endemic control groups from the village and compared with 20 healthy non endemic controls.Sandwich ELJSA was also used to measure and compare IL-10 and IFN-γin the serum of the selected groups.Results:The overall prevalence of S.haematobium was 20.0%and 0.9%in the first and the second surveys respectively,while the intensity of infection was the same in the two surveys 1.38[geometric mean egg count(GMFC)].The overall prevalence of S.mansoni infection was 68.5% and 15.4%,while the intensity of infection was 2.75(GMEC) and 1.70(GMEC) in the two surveys respectively.IgC reactivity against SWA showed no significant difference between Schistosoma positive patients and endemic controls.However,there were high significant differences between each of these two groups and the non endemic control group(P= 0,000).Schistosoma patients and exposed controls had significantly higher IL-10 concentration compared with non endemic controls.While endemic controls showed significantly higher IFN-γconcentration than patients(P = 0.000).Also there was very significant difference between IFN-γlevels of each of patients endemic controls and that of the non endemic controls(P = 0.003).Conclusions:The study concluded that IFN-γhas a role in the natural resistant to schistosoma mansoni infection. The prevalence and intensity of 5.mansoni in the Gezira Irrigation Scheme was greatly reduced.S. haematobium has disappeared from the area.

Role of GM3 ganglioside in the pathology of some progressive human diseases and prognostic importance of serum anti-GM3 antibodies

Glycosphingolipids(gangliosides)have been characterized as important biological molecules with a key role as regulators in many physiological processes on cellular,tissue,organ,and organism levels.The deviations in their normal amounts,production,and metabolism are very often related to the development of many multi-factor socially important diseases.GM3 ganglioside,as a small molecule,plays important roles in the cascade regulatory pathways in the pathology of many disorders like neurodegenerative diseases,autoimmune diseases,inflammation,diabetes,malignant transformation,and others.Ganglioside GM3 and its derivatives are membrane-bound glycosphingolipids composed of an oligosaccharide head structure containing one sialic acid residue.These molecules transduce signals involved in cell surface events,including the phosphorylation of transmembrane receptors.This ganglioside is the most widely distributed among tissues,and it serves as a precursor for most of the more complex ganglioside species.GM3 inhibits the function of fibroblast growth factor receptor,and cell growth is regulated by GM3-enriched microdomain.GM3 is thought to inhibit immunologic functions,such as the proliferation and production of cytokines by T cells.On the other hand,the anti-ganglioside antibodies(AGAs)are important in many acquired demyelinating immunemediated neuropathies,like Multiple sclerosis(MS),Guillain–Barrésyndrome(GBS)and its variation,Miller–Fisher syndrome(MFS)and could be suggested as important diagnostic and prognostic markers about the describe diseases and their etiology.We show that the complexes of anti-ganglioside antibodies to GM3(detected by ELISA)may be useful diagnostic and prognostic tool markers for autoimmune diseases,neurodegenerative disorders,malignancy,diabetes,and inflammation.Our pilot studies suggest increased serum IgG anti-GM3 antibodies titers in patients with secondary progressive MS(SPMS),throat cancer,elder people with diabetes(89–96 years),old Lewis rats(30–33 months),and in the serum of subjected on lead intoxication BALB/c mice treated by salinomycin.We observed no changes in the titers in healthy elder people(89–96 years),in 70-year-old woman on dialysis,in relapsing-remitting MS(RRMS)patients on long-term treatment with Glatiramer acetate,Laquinimod,and Interferons,as well as in 18–22 months old Wistar rats and subjected on lead intoxication BALB/c mice treated by monensin and dimercaptosuccinic acid(DMSA).Considerable decrease of serum GM3 in early MS correlate with early damage and severe destruction of the blood–brain barrier,which provides impetus to initiate early therapy.

ANALYSIS ON EPITOPES OF IGM WITH MONOCLONAL ANTI-ISOTYPIC AND ANTI-IDIOTYPIC ANTIBODIES AGAINST IgM FROM B-CLL

A double antibodies additivity ELISA test was employed to identify the epltopes which can be recognized by monoclonal antibodies (McAbs) against IgM from B chronic lymphocyte leukemia (B-CLL). The computer grouping programme analysis showed that 4 and- isotypic MaAbs could be divided into two groups and 10 anti- idiotype McAbs could be divided into four groups. The result was consistent with that of the indirect sandwich ELISA and inhibition ELISA test. It suggested that there were at least 6 distinct IgM epitopes which can react specifically with 14 McAbs. Our study indicated that the combination of the additivity ELISA test and the computer grouping programme analysis is of help in studying the relationship of the structure and function of antigen.

Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.