应用斑点免疫金渗滤法检测旋毛虫病患者血清IgG

目的　研究应用斑点免疫金渗滤法(DIGFA)快速检测旋毛虫病患者血清抗旋毛虫IgG抗体。　方法　采用旋毛虫肌肉期幼虫膜抗原,以胶体金颗粒结合的羊抗人IgG为标记抗体,以颜色深浅为判断阳性标准。　结果　纯化的旋毛虫肌肉期幼虫膜抗原与患者血清中特异性抗旋毛虫IgG抗体通过渗滤在硝酸纤维素膜上反应,10min内即可直接观察结果。在76份患者血清的检测中,阳性率为94.74%,与ELISA法的检出阳性率86.84%相比差异无显著性(χ2=2.83,P>0.05)。交叉试验与重复试验结果显示DIGFA具有较好的特异性及稳定性。　结论　DIGFA法可快速检测旋毛虫病患者血清特异性旋毛虫IgG抗体     

旋毛虫病p49抗原相关抗体的ELISA检测

目的 以纯化的融合蛋白p49/GST为抗原建立ELISA检测方法。方法 对一批试验血清进行间接ELISA检测。结果 19份工人感染鼠血清、5份人工感染猪血清、4份病人血清呈IgG抗体阳性,21份人工感染鼠血清呈IgM抗体阳性,而正常对照血清及300份屠宰场待检猪血清均呈阴性反应,其结果与常规压片法结果相符。结论 融合蛋白p49/GST对于研制旋毛虫病的诊断抗原具有潜在应用价值。     

胶体金免疫渗滤法快速检测幽门螺杆菌血清抗体的研究和应用

目的建立一种简便、快速、准确的检测胃幽门螺杆菌(H.pylori)抗体的新型血清学诊断方法。方法用胶体金标记技术与渗滤法相结合,建立了快速胶体金免疫渗滤法(DIGFA),与ELISA对比检测145份H.pylori感染血清,并与病理学检查对比检测108份H.pylori血清。做阻断试验和IgG标准品最小蛋白含量的检出观察,对其敏感性、特异性做了分析并调查了216例不同年龄组的人群感染率。结果本法与ELISA相比,两者符合率为95.1％;与病理组织染色检查比较,两者符合率为88.8％。DIGFA可检出IgG标准品的最小蛋白量为10μg     

金免疫斑点渗滤法检测日本血吸虫抗体的应用

建立了检测血吸虫病人血清抗体的金免疫斑点渗滤法（DIGFA）。以该法检测107例粪检阳性血吸虫病人血清,阳性率为96．2％,119份健康人血清均为阴性;与常规ELISA对照,符合率达99．1％,显示两法敏感性和特异性相近,但DIGFA操作更为简便快速。     

旋毛虫p49抗原基因原核表达产物的纯化

目的建立旋毛虫p49抗原基因原校表达产物的纯化方法。方法菌体经冻融、超声破菌及提取包涵体后,用离子交换和凝胶过滤层析纯化蛋白质。结果纯化后的融合蛋白P49／GST经SDS－PAGE电冰鉴定达电泳纯。双抗体夹心ELISA法检测结果表明纯化的P49／GST融合蛋白能与旋毛虫感染的鼠血清和纯化融合蛋白免疫的兔血清反应,而不与正常民和兔血清反应。结论纯化后的融合蛋白P49／GST具有较高的纯度及较强的免疫活性,可望作为旅毛虫病的免疫诊断抗原。     

金免疫斑点渗滤法检测日本血吸虫抗体的应用

建立了检测血吸虫病人血清抗体的金免疫斑点渗滤法(DIGFA)。以该法检测107份粪检阳性血吸虫病人血清,阳性率为96.2%。119份健康人血清均为阴性。与常规ELISA对照,符合率达99.1%。显示两法敏感性和特异性相近,但DIGFA操作更为简便快速。     

斑点金免疫渗滤法检测斯氏狸殖吸虫病患者血清IgG的研究

目的为寻求一种简便、快速、可靠的方法，用于诊断斯氏狸殖吸虫病。方法以斯氏狸殖吸虫成虫可溶性抗原为包被抗原，胶体金标记羊抗人IgG为显色剂，建立检测斯氏狸殖吸虫病患者血清IgG的斑点金免疫渗滤法(DIGFA)，并以dot—ELISA作平行对照。结果DIGFA和dot—ELISA分别检测91例斯氏狸殖吸虫病患者血清IgG抗体，其阳性率分别为96．7％(88／91)和92．3％(84／91)，两法差异无显著性。DIGFA分别检测健康者血清35例、日本血吸虫病患者血清25例和华支睾吸虫病患者血清22例，前者均为阴性，后两者的交叉反应率分别为4．0％(1／25)和4．5％(1／22)；DIGFA和dot—ELISA两法的总符合率达91．7％(44／48)。结论DIGFA的敏感性和特异性与dot—ELISA相近，但方法简便、快速，是一种检测斯氏狸殖吸虫病患者血清IgG抗体的较好的免疫诊断方法。     

双抗体夹心斑点金免疫渗滤法检测血吸虫循环抗原的现场应用

目的 探讨双抗体夹心斑点金免疫渗滤法（S－DIGFA）在现场的应用价值。方法 在原已建立的以抗26kDaGST基因重组蛋白多克隆抗体为捕获抗体兼测示抗体的S－DIGFA的基础上，应用该法检测血吸虫病中度流行区和传播阻断地区人群的血清共1388份，并以双抗体夹心ELISA（S－ELISA）作对照。结果 用S－DIGFA和S－ELISA平行检测血吸虫病中度流行区人群血清300份，阳性检出率分别为15．7％和17．3％；两法检测血吸虫病传播阻断地区人群血清1088份，阳性检出率分别为3．9％和3．7％。结论 S－DIGFA检测循环抗原可在现场扩大和作血清流行病学调查。     

曼氏裂头蚴可溶性抗原DIGFA法快速检测特异性抗体的潜在诊断价值

目的 探索曼氏裂头蚴可溶性抗原金标免疫渗滤法快速检测曼氏裂头蚴特异性抗体的免疫诊断价值。方法 从自然感染的青蛙中分离出曼氏裂头蚴，经口感染小鼠；于感染后第7周剖杀小鼠，收集鼠血清和虫体并计数。以曼氏裂头蚴可溶性抗原（PsA）为探针建立PsA—DIGFA，分别检测曼氏裂头蚴感染小鼠、健康小鼠、健康人群及其他寄生虫感染者血清中特异性抗体，并与常规ELISA法进行平行检测和比较。结果 DIGFA法检测感染小鼠血清中特异性抗体的敏感性为100％（49／49）、特异性为100％（30／30），与ELISA法检测结果相同；不同虫荷数感染鼠间抗体水平差异无显著性（P〉0．05），感染鼠与正常鼠之间抗体水平差异有显著性（P〈0．01）。PSA—DIGFA检测健康人群血清100人份均为阴性，检测囊虫、包虫、旋毛虫、肺吸虫、华支睾吸虫病病人血清的阳性率分别为42．5％（17／40）、10．0％（2／20）、20．0％（5／25），80．0％（8／10）、8．0％（2／25）。结论 用曼氏裂头蚴粗抗原检测感染动物血清中特异性抗体具有较好的敏感性和特异性，但与其他蠕虫感染存在较高的交叉反应。不同虫荷数感染鼠均产生较高水平抗体，组间差异无显著性。DIGFA法与ELISA法检测的敏感性和特异性相同，但前者更为简便、快速，无需特殊仪器设备，可单份检测，适合寄生虫病的临床检验和流行病学调查。若能进一步纯化检测用抗原，减少与其他蠕虫间的交叉反应，有潜在的应用价值。     

斑点金免疫渗滤法检测华支睾吸虫病患者血清抗体的研究

目的　建立一种检测华支睾吸虫病血清抗体的简便、快速、敏感和特异的新方法。方法　以华支睾吸虫成虫抗原为包被抗原 ,金标记葡萄球菌A蛋白 (SPA)为显色剂 ,建立检测华支睾吸虫病患者血清抗体的斑点金免疫渗滤法 (DIGFA) ;并以dot ELISA作平行对照。结果　DIGFA和dot ELISA分别检测 119例华支睾吸虫病患者血清抗体 ,其阳性率分别为 96 6 %和 92 4% ,两法差异无显著性。DIGFA分别检测正常人血清 40例、囊虫病患者血清 2 0例、日本血吸虫病患者血清 2 5例 ,前者均为阴性 ,后两者的交叉反应率分别为 5 %和 4%。DIGFA与dot ELISA两法的符合率达 90 9%。结论　DIGFA的敏感性和特异性与dot ELISA相近 ,且具简便、快速及不需特殊设备等优点 ,是一种检测华支睾吸虫病患者血清抗体的新方法。     

金标法检测广州管圆线虫特异性IgG抗体的研究

目的探索简易、快速的金标免疫渗滤法检测广州管圆线虫特异性抗体的诊断价值。方法以广州管圆线虫成虫可溶性抗原点样于固相硝酸纤维素膜上，以胶体金-proteinA为标记物，采用自行设计的渗滤装置，检测病人血清中广州管圆线虫特异性IgG抗体。结果检测21人份广州管圆线虫病人血清，检出阳性19份，敏感性为90．5％（19／21）；100人份健康人群血清2份阳性，假阳性率为2％（2／100）；与旋毛虫病人、血吸虫病人血清交叉反应率分别为26．7％（8／30）、3．3％（1／30），未发现与肺吸虫、囊虫、华支睾吸虫、肺结核病人血清有交叉反应。结论金标免疫渗滤法快速检测血清中广州管圆线虫特异性IgG不仅操作简便、快速，结果判读容易，不需特殊仪器设备，而且敏感性高，有较好的辅助诊断价值，与旋毛虫病人血清存在交叉反应。     

旋毛虫p49抗原相关抗体的快速检测

目的　建立旋毛虫病的快速检测技术。方法　基因工程抗原包被有色乳胶颗粒,抗抗体包被磁性颗粒,在抗体存在下形成抗原－ 乳胶－ 抗体－ 抗抗体－ 磁性颗粒的复合场,在磁场作用下沉淀下来,从而达到快速检测旋毛虫相关抗体的目的。结果　１９ 份人工感染鼠血清、５ 份人工感染猪血清、３ 份旋毛虫病猪血清、４ 份病人血清均呈阳性反应,而对照血清均为阴性反应;该方法在鼠感染旋毛虫后第五天可测出ＩｇＭ 抗体,第九天可测出ＩｇＧ抗体。结论　本检测技术简便易行,不需专用设备,可望成为一种快速诊断旋毛虫病的有效方法。     

金标渗滤法快速检测曼氏裂头蚴病患者血清IgG

目的探索简易、快速的人曼氏裂头蚴病血清学免疫诊断方法。方法将曼氏裂头蚴可溶性抗原点样于硝酸纤维素膜上,以胶体金-SPA为检测标记物,采用垂直流渗滤装置检测病人血清中曼氏裂头蚴特异性IgG抗体。结果曼氏裂头蚴病病人血清阳性检出率为92.0%(23/25),健康人群血清假阳性率为2.0%(2/100);与囊虫病、肺吸虫病、血吸虫病、华支睾吸虫病、旋毛虫病病人血清的交叉反应率分别为40.0%(16/40)、52.0%(13/25)、13.3%(4/30)、8.0%(2/25)、6.7%(2/30),未发现与广州管圆线虫病、结核病病人血清有交叉反应;与ELISA平行检测74人份血清,两种方法检测结果的一致性有极显著性意义(К=0.871,P<0.001)。结论金标渗滤法检测曼氏裂头蚴病病人血清中特异性IgG,不仅简易、快速而且检出率高,但与肺吸虫病、囊虫病病人血清存在较高的交叉反应,需根据病人饮食史等流行病学资料进行鉴别诊断。     

The usefulness of ELISA test for early serological detection of Trichinella spp. infection in pigs

Trichinellosis is a parasitic zoonosis transmitted to humans through consumption of raw or undercooked meat from animals infected with nematodes of the Trichinella genus. Every year seropositive cases are found among the human population and thus trichinellosis still remains an epidemiologically important disease. The first step of the study was the optimization of a new ELISA method enabling an early and specific serological diagnosis of trichinellosis in pigs and wild boars using excretory-secretory (ES) antigens obtained from in vitro cultures of L1 T. spiralis. Serum samples were assayed for anti-T. spiralis IgG antibodies using the new ELISA protocol and a reference test--Standard manufactured by Institut Pourquier. The optimization involved the selection of suitable plates for antigen coating, dilution of sera and antibodies and their time of incubation. On the basis of the optimization a new ELISA procedure for the detection of IgG and IgM against T. spiralis was elaborated. Conventional, Iberian pigs and SPF (Specific Pathogen Free) pigs were infected with 200, 1000 and 20,000 muscle larvae of T. spiralis. Serum samples were obtained at 5 and 1 dbi (day before infection), and 5, 10, 15, 20, 25, 30, 40, 50, 60 dpi (day post infection) and screened for specific IgG antibodies against excretory-secretory L1 T. spiralis antigens. Serum samples were obtained from the EU project Trichiporse: "Safe pork and horse meat on EU markets: early and unbiased diagnostic tests for Trichinella". Field samples of conventional pigs (1474) and wild boars (1784) were obtained from slaughter houses in different parts of Poland. Pigs were examined for the presence of Trichinella spp. using the artificial digestion method. Only four pigs were naturally infected with T. spiralis, the remaining were Trichinella larvae free. ELISA was used to examine IgG levels against L1 T. spiralis in pig and wild boar sera. The usefulness of ELISA for anti-IgG detection in pigs is usually limited by the nature of the antigen. The antigens were prepared in different laboratories: in Germany--Ag ES L1 T. spiralis (N), Italy--Ag ES L1 T. spiralis (W)) and in Poland--Ag ES L1 T. spiralis. Cut-off values for ELISA along with the estimated sensitivity and specificity were calculated using different methods: S/P%, M+3SD and ROC (Receiver Operating Characteristic). In SPF and Iberian pigs inoculated with 200, 1000 and 20,000 L1 T. spiralis, specific antibodies were detected 40, 30 and 25 dpi, respectively, with the use of the Standard (reference test). The analysis of the two ELISA procedures demonstrated a high sensitivity and specificity for the newly elaborated test utilizing the Ag ES L1 T. spiralis. In conventional pigs infected with 20,000 L1 T. spiralis specific antibodies were detected from 20 dpi when employing the new protocol. Similar results for the Standard and new ELISA test were obtained for serum samples of conventional pigs infected with 200 and 1000 larvae, which became positive from 40 dpi and 30 dpi, respectively. The results showed that both: the Standard and new protocols were comparable, and based on this, the new test was applied for further research. Results obtained adopting the new protocol with three antigens showed that two of them: Ag ES L1 T. spiralis (W) and Ag ES L1 T. spiralis are similar. The specific IgG antibodies for infective doses of 200 and 1000 larvae for these antigens were detectable 40 and 30 dpi respectively. In pigs infected with the highest dose of T. spiralis larvae IgG antibodies were detectable from 20 dpi when Ag ES L1 T. spiralis was used. These results strongly indicate that in examined pigs, the specific IgG response to T. spiralis infection is dose dependant. Of 1474 examined pig sera only 0.99% gave a positive signal against ES L1 T. spiralis antigen. Of 1784 examined wild boars sera only 0.68 % gave positive results using the new ELISA protocol. ELISA is a useful method for detecting specific IgG antibodies in pigs experimentally infected with different doses of T. spiralis and naturally infected pigs. In pigs the specific IgG response is dose dependant. The Ag ES L1 T. spiralis increases the specifity of the method and reduces false positive results. Simultaneous use of both methods: digestion and ELISA for the diagnosis of Trichinella in naturally infected pigs and wild boars may increase the chances of eliminating meat infected with T. spiralis larvae.     

抗旋毛虫肌肉期幼虫排泄分泌物中46-58kDa蛋白IgM和IgG抗体的研究

目的寻求简便、快速、可靠的方法,用于旋毛虫病的诊断及疗效考核。方法建立检测抗旋毛虫肌肉期幼虫排泄分泌物(TsL1-ES)中46-58kDa蛋白抗体的斑点金免疫渗滤试验(DIGFA),并对患者在治疗前和治疗后血清中特异抗体水平的动态变化进行研究。结果旋毛虫病患者治疗前血清中特异IgM和IgG抗体的检出率分别为94.83%(55/58)和91.38%(53/58);正常人血清(100份)、囊虫病(25份)、血吸虫病(20份)、肺吸虫病(20份)及蛔虫病(22份)病人血清均未检出该两类特异性抗体。52例两类抗体均阳性的旋毛虫病患者经有效药物治疗后3个月、6个月、1年、1.5年连续采血,经DIGFA检测连续观察的结果表明,IgM抗体出现较早,阴转较为明显和迅速,治疗后3月的阴转率达46.15%(24/52),1年的阴转率可达88.46%(46/52);而IgG抗体则出现较晚,阴转缓慢,治疗后1.5年的阴转率仅为23.08%(12/52)。结论DIGFA为检测抗TsL1-ES中46-58kDa蛋白抗体的有效方法,检测IgM抗体可用于诊断及疗效考核,而IgG抗体的检测不宜用来判断疗效。     

旋毛虫编码新生幼虫p46 kDa抗原基因重组融合蛋白对小鼠的免疫保护性研究

目的 探讨旋毛虫编码新生幼虫p46 kDa抗原基因重组融合蛋白WN10对小鼠的免疫保护性。方法 将纯化的重组融合蛋白WN10以20μg/只的剂量分3次免疫小鼠后，攻击感染旋毛虫肌幼虫200条／只，检查旋毛虫7日龄成虫数、雌虫体外产生新生幼虫数、感染35d的肌幼虫数并计算减虫率，同时用ELISA法检测血清中抗WN10抗体IgG滴度。结果WN10免疫小鼠后获得旋毛虫7日龄成虫、肌幼虫的减虫率分别为64．28％和61．21％，均与感染对照组和佐剂对照组差异显著；获得雌虫产新生幼虫的减虫率为16．46％，与感染对照组和佐剂对照组差异不显著；WN10免疫组小鼠在第3次免疫后1周可检测到高滴度的抗WN10抗体IgG，在攻击感染旋毛虫9周后仍维持在较高水平。结论 编码旋毛虫新生幼虫p46 kDa抗原基因重组融合蛋白可诱导小鼠产生较强的抗旋毛虫保护性免疫。     

Activity of specific IgG, IgM and IgE antibodies in human trichinellosis.

The activity of IgG, IgM and IgE antibodies to somatic antigen of Trichinella spiralis in the sera of patients with trichinellosis at various intervals after infection was examined by means of ELISA. Mathematical analysis of the dose-response curves was used. Elevated level of IgG and IgM antibodies of relatively high avidity and of rather low IgE avidity was documented. Amount or avidity of IgG antibodies was found to be most useful for the diagnosis of trichinellosis (85% positive results in patients' sera). The isotype of IgM avidity constitutes a better diagnostic value than the amount of it (60% and 35% of positive results, respectively).