日本血吸虫成虫抗原和虫卵抗原检测先天性感染仔兔血清抗体的动态变化

目的　观察仔兔血清抗体动态变化 ,探讨家兔日本血吸虫病垂直传播的体液免疫反应规律。方法　 5只怀孕晚期母兔分别人工感染 5 0 0条日本血吸虫尾蚴 只 ,仔兔出生后 4 3d起每隔 2周采血收集血清 ,至仔兔发育成熟 (约出生后113d)。分别运用日本血吸虫成虫抗原 (AWA)和可溶性虫卵抗原 (SEA) ,ELISA法检测血清中特异性IgG、IgM抗体 ,观察动态变化。结果　仔兔先天性感染率为 6 0 % (12 2 0 ) ,其中 5只双性感染 ,7只单性感染。 2 0只仔兔 ,抗AWA IgG、抗AWA IgM抗体检测始终呈阴性 ;抗SEA IgG检测 ,5只双性感染仔兔有 4只于出生后 5 7d起陆续呈阳性 ,其余 16只始终为阴性 ;2 0只仔兔抗SEA IgM也始终呈阴性。结论　先天性感染日本血吸虫病的仔兔呈低免疫应答状态 ,可能存在免疫耐受     

先天性感染日本血吸虫家兔攻击感染后血清抗体动态的观察

日本血吸虫抗原系统相对比较复杂，汤益等对先天性感染日本血吸虫仔兔于出生后第55天攻击感染，以ELISA法检测仔兔血清特异性针对可溶性虫卵抗原(SEA)抗体，结果仔兔对攻击性感染呈低反应状态。本研究采用ELISA法检测先天性感染仔兔攻击感染后血清针对日本血吸虫抗原(AWA)IgC、IgM抗体并观察其动态变化，同时重复SEA-ELISA法检测结果，进一步探讨日本血吸虫病先天性感染仔兔的体液免疫应答。     

血吸虫病再感染易感性相关抗原的免疫学鉴定

采用Western blot技术．用再感染易感组血清和拮抗组血清对AWA进行免疫识别．为寻找评价人群再感染易感性的诊断抗原和疫苗候选分子提供有价值的信息。结果显示，拮抗组IgG对36kDs和拮抗组IgE对69kDa处条带强烈识别；易感组IgG4对47、32和25kDa处条带强烈识别。结果提示，36kDa和69kDa可能与诱导机体产生针对再感染抗力的免疫应答有关，47、32和25kDa可能与“封闭性抗体”产生有关。     

日本血吸虫成虫67kDa分子抗原的诊断价值

为检测日本血吸虫成虫67kDa分子抗原（sjAWA67)对血吸虫病的诊断价值。本研究通过SDS-PAGE和电渗方法,从日本血吸虫成虫抗原中分离纯化出67kDa分子抗原,并用该抗原包被酶标反应板微孔。进行常规ELISA检测。结果对急,慢性血吸虫病患血清的检出率分别为100%和95%,与正常人血清,肝吸虫病和肺吸虫病患血清均未出现明显的变叉反应,44例血吸虫病患治疗后3、6和12个月后的阴转率分别达45.7%,75.0%和90.09%。表明SjAWA67蛋白具有较好的疗效考核价值和现场应用前景。     

日本血吸虫未成熟虫卵可溶性抗原诊断血吸虫病的初步探讨

用日本血吸虫未成熟虫卵可溶性抗原(SIEA)作探针，采用ELISA和ELIB检测血吸虫病人血清抗体，对急慢性血吸虫病人的控出率分别为100％和98．2％，未出现假阳性及交叉阳性反应；治疗后3、6和12个月血清抗体的阴转率分别为32．3％、50．9％和64％。其中抗82，73，67，52，42，38，32，31，28，26．21，18kDa分子的血清抗体消失较快。结果表明，SIEA在血吸虫病的诊断中具有较高的敏感性、特异性和潜在的疗效考核价值。     

单克隆抗体识别日本血吸虫循环虫卵抗原表位的初步研究

本文采用免疫印迹试验对抗日本血吸虫卵可溶性糖蛋白单克隆抗体SM_(21-2)识别感染兔血清中循环虫卵抗原表位作了初步的分析。将Dot-ELISA检测呈阳性反应的感染兔血清进行SDS凝胶电泳,再转移至NC膜上,采用间接ELISA染色。结果发现SM_(21-3)能识别感染10、100、1000条尾蚴兔血清中27kDa的循环虫卵抗原表位,而正常血清无此区带。同时发现在46kDa处存在非特异性反应。不同的封闭剂对印迹效果有一定的影响,用0.3％Tween-PBS获得了较好的结果。     

高效天然分子疫苗免疫猪血清筛选尾蚴抗原的结果分析

采用日本血吸虫天然分子(Schistosoma japonicum immature egg ws-vaccine,SjiEw)免疫猪血清识别日本血吸虫可溶性尾蚴抗原(Soluble cercariae antigen,SCA),以筛选新的具有保护性免疫潜在价值的抗原分子。按常规方法制备日本血吸虫尾蚴抗原。家猪随机分组并分次免疫日本血吸虫天然分子疫苗,制备SjiEw疫苗免疫猪血清,通过蛋白免疫印迹技术分析其与SCA的免疫反应性。结果显示SDS-PAGE中SCA有16条主带。SjiEw疫苗免疫猪血清共筛选6种具免疫学活性的抗原分子,分子量分别为:50、56、66、68、70、85kDa。这些抗原分子的获得为以后的天然分子编码基因工程疫苗的制备奠定基础。     

用重组SAG1抗原检测弓形虫抗体

目的探讨重组SAG1抗原对弓形虫IgG和IgM抗体的检测效果。方法用rSAG1作抗原建立免疫印迹方法（rSAG1-WB）,与玻片虫体过氧化物酶免疫染色试验（TSHE）平行检测不同来源血清。结果15例病原学检查阳性小鼠血清和5例免疫兔血清的IgG抗体均为阳性,30例正常小鼠血清和10例正常兔血清均未出现阳性反应。rSAG1-WB检测可疑弓形虫病患者血清阳性率为60．3％（38／63）,献血员血清阳性率为6％（3／50）,与TSHE检测结果（65．1％和4％）均无统计学差异（P〉0．05）。1例IgM强阳性血清和13例IgM弱阳性血清在Western—blot检测中分别出现相应的强阳性与弱阳性反应,50例献血员血清均未出现IgM阳性反应,结果与TSHE一致。结论rSAG1-WB检测弓形虫IgG和IgM抗体均具有高度的敏感性和特异性．与TSHE的符合率高。     

抗兔μ链和抗兔γ链血清的制备及初步应用

用常规生化方法提取正常兔血清中的IgM,用吸附法制备抗兔μ链血清。免疫电泳证明所制备的抗兔μ链血清与兔全血清仅有一条沉淀线,与兔IgG、IgA没有交叉反应。与抗兔μ链结合的兔血清成份有抗体活性,对2-巯基乙醇敏感;分子排阻层析证明,分子量与入骨髓瘤IgM相同。同时制备了抗兔Υ链血清。应用这二种血清检测经福氏2a志贺氏免疫兔的血清抗体证明它们分别对IgM、IgG有特异性,可用于观察免疫后特异IgM、lgG抗体产生的动态规律。     

固相酶联免疫测定法检测NS1抗原在登革病毒感染早期诊断中的应用

目的探讨固相酶联免疫测定（ELISA）法检测NS1抗原在登革病毒感染早期诊断中的应用价值。方法选取登革病毒感染早期患者血清171份,非登革病毒感染发热患者血清11份,正常人血清10份,采用ELISA法检测全部192份血清的登革病毒NS1抗原和IgM抗体;采用逆转录-聚合酶链反应-限制性内切酶酶切片段长度多态性分析（RT-PCR-RFLP）技术对发病5 d内的125份血清进行扩增和鉴定分型;并采用C6/36细胞微量培养法对发病第1、2天的41份血清进行登革病毒分离培养。结果登革病毒感染患者发病2 d内、3～5 d以及6～10 d血清NS1抗原的检出率分别是92.7%（38/41）、83.3%（70/84）、10.9%（5/46）;IgM抗体的检出率分别是2.4%（1/41）、51.2%（43/84）、97.8%（45/46）;非登革病毒感染的发热患者及正常人血清中,有1例疟疾患者血清登革病毒IgM抗体呈阳性,NS1抗原无一例阳性。RT-PCR在登革病毒感染患者发病第1、2天和3～5天的检出率分别是85.4%（35/41）、83.3%（70/84）;登革病毒感染患者发病第1、2天血清的病毒分离培养阳性率分别是80.0%（16/20）、38.1%（8/21）,总分离率58.5%（24/41）;RT-PCR-RFLP分型鉴定技术及间接免疫荧光法（IFA）均证实2006年广州流行株为登革Ⅰ型病毒。结论ELISA法检测登革病毒NS1抗原操作技术成熟,且具有敏感性高、特异性好的特点,对登革病毒感染的早期诊断和疫情的早期控制具有重要意义,适合于基层医疗机构常规应用。     

Differences in prevalence,intensity of infection and parasite-specific antibody levels do not predict different age-infection profiles

Acquired immunity is believed to influence the age-infection profile of Schistosoma infections. We compared antibody responses against Schistosoma mansoni adult worm antigen (AWA) and soluble egg antigen (SEA) in 164 residents of two communities with different levels of infection. IgG, IgA, IgM, IgE, and IgG subclass 1 to 4 antibodies were determined by ELISA. Seventy-five of the subjects were from Harbu, an area with a prevalence of 39% and an intensity of infection of 116 eggs per gram of stool (EPG), whereas 89 subjects were from Bati, with a prevalence of 66% and intensity of infection of 256 EPG. In both communities the prevalence and the intensity of infection were highest in the age group 10-14 years, although both were significantly higher in Bati than in Harbu. Mean levels of AWA-specific IgA, IgM, IgG, IgG1 and IgG2, and of SEA-specific IgG, IgM, IgG2 and IgG3 were significantly higher in Bati than in Harbu. However, mean levels of IgE against worm and egg antigens were significantly higher in Harbu than in Bati. Significant differences were detected in the levels of IgA, IgE, IgG, IgM, IgG1 and IgG2 against AWA, and in IgE, IgM, IgG2 and IgG3 against SEA according to the place of residence. The levels of anti-AWA IgG, IgG1 and IgG2 and anti-SEA IgG, IgG1 and IgG4 were significantly associated with the intensity of infection. Anti-AWA IgM levels were associated with age, whereas sex and age had interacting effects on the levels of AWA-specific IgG1 and SEA-specific IgG and IgM. Antibody responses exhibited different age-related patterns in the two communities. This may indicate that differences in history of exposure influence the evolution of immune responses. However, the study did not support the view that differences in antibody levels between communities subject to different levels of infection result in a systematic deviation in age-infection profile (the "peak shift").     

Sera of Schistosoma japonicum-infected patients cross-react with diagnostic 31/32 kD proteins of S. mansoni.

Schistosoma mansoni adult worm antigens were tested for cross-reactions with sera obtained from patients infected with S. japonicum. The sera consistently recognized a doublet of bands, in immunoblots, which had molecular weights of approximately 31 and 32 kilodaltons (kD). This reaction was found to be markedly reduced with sera of patients who had received chemotherapy and who had a low risk of reinfection. Sera obtained from uninfected persons or from patients infected with other parasites never reacted with the antigen doublet. Schistosoma japonicum-infected mice produced antibodies during prepatency which predominantly recognized antigens of this molecular weight range in immunoblots performed with S. mansoni or S. japonicum proteins. Sera from S. mansoni-infected patients with a high specificity for the diagnostic S. mansoni-antigen cross-reacted with a corresponding component also in S. japonicum worms. Immunofluorescence assays performed with sera of schistosomiasis japonica patients confirmed earlier results localizing the diagnostic 31/32 kD antigens in the gut of S. mansoni. These cross-reacting 31/32 kD S. mansoni protein antigens may be applied for the immunodiagnosis of schistosomiasis japonica.     

检测日本血吸虫循环抗原的压电免疫传感器中抗体的纯化与固定

采用饱和硫酸铵沉淀法及凝胶过滤法从感染兔血清 (InRS)和免疫兔血清 (ImRS)中纯化出IgG抗体 ,分别经过SDS PAGE鉴定其纯度及对流免疫试验、免疫双扩散试验鉴定其活性和效价 ,以便制成一种新型的液相压电免疫传感器 ,用于检测兔与病人血清中的日本血吸虫循环抗原 (SjCAg)。初步应用该法检测了不同感染度的兔血清 ,发现频移值变化在一定范围内与感染度成正比。     

Immunoblot detection of antibodies to Toscana virus

Sera from patients with sandfly fever caused by Toscana virus (TOSV) infection were tested by immunoblot for specific antibody response to TOSV derived from infected Vero-E6 cells. The 28 kDa TOSV nucleoprotein (N) was identified as the major immunodominant protein recognized by immunoblot. In sera of patients with acute TOSV infection, specific antibodies of the IgM, IgA, and IgG class were detected. Using sandfly fever virus, serotypes Sicilian (SFSV) and Naples (SFNV), as antigens for immunoblot, TOSV antibody-positive sera cross-reacted with the corresponding N proteins. These sera reacted for IgM and IgG by SFSV immunoblot, and for IgM by SFNV immunoblot. The diagnosis of sandfly fever may be confirmed by TOSV immunoblot. © 1996 Wiley-Liss, Inc.     

抗日本血吸虫SEA特异性IgY应用于循环抗原检测的研究

本研究应用抗日本血吸虫可溶性虫卵抗原(SEA)的鸡卵黄免疫球蛋白(IgY)建立敏感、特异的检测循环抗原的双抗体夹心酶联免疫吸附试验( ELISA).用SEA皮下多点注射法免疫海蓝鸡,水稀释法制备IgY抗体,以辣根过氧化物酶标记纯化的IgY抗体(IgY-E)和兔抗IgG抗体(IgG-E)分别作为检测抗体,IgY抗体和兔抗...     

Use of recombinant calreticulin and cercarial transformation fluid (CTF) in the serodiagnosis of Schistosoma mansoni

Schistosomiasis is traditionally diagnosed by microscopic detection of ova in stool samples, but this method is labour intensive and its sensitivity is limited by low and variable egg secretion in many patients. An alternative is an ELISA using Schistosoma mansoni soluble egg antigen (SEA) to detect anti-schistosome antibody in patient samples. SEA is a good diagnostic marker in non-endemic regions but is of limited value in endemic regions, mainly because of its high cost and limited specificity. Here we assess seven novel antigens for the detection of S. mansoni antibody in an endemic region (the Northern Nile Delta). Using recombinant S. mansoni calreticulin (CRT) and fragments thereof, anti-CRT antibodies were detected in the majority of 97 patients sera. The diagnostic value of some of these antigens was, however, limited by the presence of cross-reacting antibody in the healthy controls, even those recruited in non-endemic areas. Cercarial transformation fluid (CTF), a supernatant that contains soluble material released by the cercariae upon transformation to the schistosomula, is cheaper and easier to produce than SEA. An ELISA using CTF as the detection antigen had a sensitivity of 89.7% and an estimated specificity of 100% when used in non-endemic regions, matching the performance of the established SEA ELISA. CTF was substantially more specific than SEA for diagnosis in the endemic region, and less susceptible than SEA to cross-reacting antibody in the sera of controls with other protozoan and metazoan infections.     

犬蠕形螨病诊断抗原制备研究

通过刮取重症病犬蠕形螨发病部位的皮屑 ,用 5 %NaOH消化 2h后进行虫体浓集 ,将浓集的虫体冻融、研磨、超声破碎 ,经 80 0 0r min离心 2 0min ,取上清为蠕形螨盐溶性粗抗原 ;将沉渣用尿素混匀 ,再次超声 ,经 80 0 0r min离心 2 0min ,取上清为蠕形螨尿素溶性粗抗原。所得抗原用Bradford法进行蛋白浓度检测 ,浓度分别为 1 4mg mL和 1 1mg mL。经过葡聚糖凝胶层析 (G 10 0 ) ,分别得到两个层析峰。经SDS 聚丙烯酰胺凝胶电泳 ,盐溶性粗抗原显示 2条主蛋白带 ,分子量约为 4 5 7kDa和 2 7 5kDa ;尿素溶性粗抗原显示2条主蛋白带 ,分子量约为 4 6 8kDa和 2 7 5kDa ,用正常犬皮处理的对照抗原却出现至少 9条蛋白带 ,分子量范围为 :2 7kDa～ 110kDa。将抗原稀释至 1∶30 0 ,血清按 1∶30 0稀释 ,酶标SPA (葡萄球菌A蛋白 )作为二抗 ,通过ELISA检测 ,尿素溶性粗抗原层析峰第一峰具有较好抗原性 ,实际检测 12例临床检查发现蠕形螨的临床病犬和 19例未发现蠕形螨的临床健康犬 ,阳性符合率和阴性符合率分别为 91 6 7%和 78 95 %。经与其他皮肤病犬进行检测发现该抗原能较好地与其他皮肤病进行鉴别 ,与猪蠕形螨阳性血清有一定的交叉反应。获得了敏感性和特异性较高的犬蠕形螨病诊断抗原     

Kinetics and magnitude of antibody responses against the conserved 47-kilodalton antigen and the variable 56-kilodalton antigen in scrub typhus patients

Western blot analysis of Orientia tsutsugamushi whole-cell lysates with scrub typhus patient sera has identified at least five protein antigens of O. tsutsugamushi with molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.