检测鼠疫抗体胶体金免疫层析试纸条的研制

目的建立快速、简易的检测鼠疫F1抗体的胶体金免疫层析法(G ICA)。方法(1)采用胶体金颗粒标记纯化F1抗原,并将标记物喷于玻璃纤维;同时将纯化F1抗原喷线固定于硝酸纤维素膜上,用于F1抗体的捕捉;按常规组装成检测鼠疫抗体免疫层析试纸条。(2)采用该试纸条与血凝法对同一份兔抗F1抗体进行检测,以评价该试纸条的敏感性。(3)采用该试纸条对44株非鼠疫菌的免疫鼠血清进行检测,以评价该试纸条的特异性。(4)采用该试纸条、血凝法及ELISA对607份血清标本进行检测,以评价该试纸条对现场材料的检测效果。结果(1)该试纸条可在15 m in之内完成检测;(2)在敏感性上,该试纸条对同一份免疫兔血清的检测较血凝法高一个滴度;(3)对被试的44株所选菌株的免疫鼠血清的检测均为阴性;(4)在对607份血清标本的检测中,免疫层析试纸条、血凝及ELISA三种方法的符合率中度,而免疫层析试纸条的敏感性分别比血凝与ELISA高111%和90%。结论以纯化的鼠疫F1抗原为基础建立的G ICA检测鼠疫F1抗体的方法特异性强、灵敏度高、简便快速,无需特殊仪器设备,有较大的推广应用价值。     

快速诊断家畜日本血吸虫病免疫层析试纸条的研制与初步应用

目的开发研制敏感、简便、快速、经济的日本血吸虫病诊断试纸条。方法利用双抗原夹心法,以日本血吸虫可溶性虫卵抗原(SEA)为检测抗原,以胶体金标记SEA为探针,采用自行设计的免疫层析试纸条装置,检测家畜血清中血吸虫特异性IgG抗体,并用ELISA方法进行比较。结果用该试纸条检测107份人工感染血吸虫病羊血清,其检出率为91.6%;检测80份健康绵羊血清,阴性符合率为87.5%;检测20份粪检阳性水牛血清及15份粪检阴性血清,其阳性符合率为100.0%,阴性符合率为86.7%;检测24份肝片形吸虫病羊血清,交叉反应率为12.5%,与锥虫病牛血清未见交叉反应;与ELISA检测结果比较,两种方法具有良好的一致性。结论应用试纸条诊断家畜日本血吸虫病敏感性高、特异性强,且操作简便、快速,不需特殊仪器设备,适合于基层使用。     

胶体金免疫层析试纸条对云南鼠疫现场材料的检测

目的评价检测鼠疫抗原及抗体胶体金免疫层析试纸条(G ICA及RG ICA)对现场材料的检测效果。方法采用G ICA分别对采自云南省的607份血清标本(查鼠疫抗体)及572份鼠脏器标本(查鼠疫抗原)进行检测,同时以血凝试验(IHA及R IHA)与酶联试验(ELISA及F1-ELISA)作为对照。结果(1)在对607份血清标本的检测中,G ICA、IHA及ELISA三种方法的符合率中度,而G ICA的敏感性比IHA与ELISA分别高111%和90%;(2)在对572份鼠脏器标本的检测中,RG ICA、R IHA及F1-ELISA三种方法的符合率中度,而RG ICA的敏感性比R IHA与F1-ELISA分别高100%和14.3%。结论检测鼠疫的G ICA及RG ICA特异性强、灵敏度高、简便快速,有较大的推广应用价值。     

人卡他莫拉单克隆抗体胶体金试纸的制备及初步应用

目的制备抗UspA1蛋白单克隆抗体,研制能快速、灵敏、准确检测卡他莫拉菌的胶体金免疫层析试纸,以期用于人卡他莫拉菌的临床检测。方法将重组UspA1-His蛋白作为抗原,免疫小鼠,并制备单克隆抗体;通过Western blot技术以及免疫荧光技术鉴定抗体的特异性;以双抗体夹心的原理研制胶体金免疫层析检测试纸条,并对其特异性、敏感性及稳定性进行评价分析。结果筛选出两株分泌特异性抗体的杂交瘤细胞株,经腹水制备纯化得到单克隆抗体,并验证了两种抗体均能特异性识别天然UspA1蛋白;利用双抗夹心原理制备的胶体金试纸能在10 min内快速检测卡他莫拉菌,检测灵敏度高(1×10~5 CFU/ml)、特异性强,与其他常见9种的呼吸道病原菌诸如肺炎链球菌、流感嗜血杆菌、肺炎支原体、嗜肺军团菌等无交叉反应;试纸条在37℃保存有良好的重复性和稳定性;200份临床样品检测结果与平板培养法的阳性符合率为93.3%。结论本研究制备了特异性强的抗UspA1蛋白单克隆抗体;以此研制的胶体金免疫层析试纸可快速、简便、灵敏的检测卡他莫拉菌,适用于呼吸道感染的临床快速检测。     

诊断家畜日本血吸虫感染胶体金免疫层析试纸条的研制

目的 目的 研制一种可用于疫区现场快速诊断家畜日本血吸虫感染的胶体金免疫层析试纸条。方法 方法 设计重组蛋 白G并在大肠杆菌中表达, 得到重组蛋白。对重组蛋白G进行胶体金标记并制备金标垫, 分别用可溶性虫卵抗原 （SEA） 和重组蛋白G划线, 作为检测线和质控线。采用组装的试纸条检测标准阴、 阳性BALB/c鼠与新西兰大白兔血清, 以及粪 检阴、 阳性的绵羊与水牛血清, 评估其灵敏度和特异度; 利用组装的试纸条检测前后盘吸虫病病牛血清, 评价其交叉反应 性。结果 结果 成功构建了重组蛋白G的原核表达质粒并在大肠杆菌中表达。以重组蛋白G制备的试纸条可用于检测日本 血吸虫感染BALB/c鼠、 新西兰大白兔、 水牛、 绵羊血清抗体。该试纸条检测日本血吸虫感染小鼠与兔血清的灵敏度、 特 异度均为100%; 检测绵羊血清的灵敏度为100%, 特异度为88.46%; 检测水牛血清的灵敏度为94.44%, 特异度为100%; 其与前后盘吸虫交叉反应率为5.88%。结论 结论 成功研制能检测多种家畜血吸虫病的胶体金免疫层析试纸条, 其检测家 畜血吸虫感染的灵敏度和特异度均较高。     

类鼻疽病快速诊断的临床应用

目的类鼻疽病快速诊断临床上的应用效果。方法使用2000hP片段的特异抗原作间接ELISA包被抗原对临床上疑为类鼻疽患者进行血清学检测;同步进行类鼻疽假单胞菌培养，结合X线、实验室检查及治疗效果进行诊断。结果在1808份标本中阳性70人份，诊断过去感染11人，现症感染21人，细菌培养阳性17人。结论该快速试验，诊断有效率达98.8％，漏诊率3.9％，误诊率为l％，完全适合于临床患者的快速诊断。     

猪带绦虫六钩蚴TSOL18重组蛋白快速诊断试纸条研制

目的以猪带绦虫六钩蚴重组蛋白(TSOL18)为抗原,建立一种简便快速的猪囊虫抗体检测方法,评价其血清学诊断的价值。方法用胶体金颗粒标记纯化的TSOL18重组蛋白,将兔抗TSOL18-GST-IgG和TSOL18重组抗原分别喷涂于硝酸纤维素膜的质控线和检测线上,与PVC垫板等部件按顺序装配成猪囊虫抗体快速检测试纸条;用该试纸条检测猪血清样品测定敏感性和特异性。结果该试纸条与全囊虫抗原ELISA的相对敏感性和特异性分别为75.0%(12/16)和95.2%(40/42),与剖检计数法的相对敏感性达80.0%(12/15)。试纸条在4℃存放12个月,检测结果稳定,重复性好。结论基于TSOL18建立的胶体金免疫层析试纸条操作简单、快速敏感、稳定性好,可用于猪囊虫病感染的诊断和筛查。     

免疫层析试纸条检测感染小鼠肉汁中抗旋毛虫抗体的研究

目的 建立一种肉类旋毛虫感染的快速免疫学检测方法.方法 以胶体金标SPA和旋毛虫肌幼虫ES抗原制备免疫层析试纸条,对不同剂量旋毛虫幼虫感染小鼠后不同时间的血清及肉汁抗体进行检测,并与ELISA、镜检法及消化法的检测结果进行比较.结果 用100、300、500条旋毛虫幼虫感染小鼠后6周,3组小鼠的血清及肉汁抗体阳性率均达100%;试纸条法对血清与肉汁的抗体检出率差异无统计学意义(χ2=0.22, P＞0.05).试纸条法和ELISA检测100条幼虫感染小鼠肉汁的阳性率分别为91.3%和100%(χ2=2.09, P＞0.05),检测300、500条幼虫感染小鼠肉汁的阳性率均为100%;试纸条法和镜检法对3组小鼠感染后5～7周的肉汁和膈肌检测的阳性率均为98.6%.消化法检查每克肌肉虫荷＜33条幼虫的6份标本,试纸条5份阳性,每克肌肉中含有6条幼虫时可被试纸条法检出.感染旋毛虫小鼠肌肉4 ℃保存1～7 d及-20 ℃保存1～7个月的肉汁抗体阳性率均为100%.结论 免疫层析试纸条可用于新鲜肉、冷藏肉及冷冻肉中抗旋毛虫抗体的检测.     

免疫层析试纸条检测感染小鼠肉汁中抗旋毛虫抗体的研究

目的建立一种肉类旋毛虫感染的快速免疫学检测方法。方法以胶体金标SPA和旋毛虫肌幼虫ES抗原制备免疫层析试纸条,对不同剂量旋毛虫幼虫感染小鼠后不同时间的血清及肉汁抗体进行检测,并与ELISA、镜检法及消化法的检测结果进行比较。结果用100、300、500条旋毛虫幼虫感染小鼠后6周,3组小鼠的血清及肉汁抗体阳性率均达100%;试纸条法对血清与肉汁的抗体检出率差异无统计学意义（χ2=0.22,P〉0.05）。试纸条法和ELISA检测100条幼虫感染小鼠肉汁的阳性率分别为91.3%和100%（χ2=2.09,P〉0.05）,检测300、500条幼虫感染小鼠肉汁的阳性率均为100%;试纸条法和镜检法对3组小鼠感染后5-7周的肉汁和膈肌检测的阳性率均为98.6%。消化法检查每克肌肉虫荷〈33条幼虫的6份标本,试纸条5份阳性,每克肌肉中含有6条幼虫时可被试纸条法检出。感染旋毛虫小鼠肌肉4℃保存1-7 d及-20℃保存1-7个月的肉汁抗体阳性率均为100%。结论免疫层析试纸条可用于新鲜肉、冷藏肉及冷冻肉中抗旋毛虫抗体的检测。     

类鼻疽伯克霍氏德氏菌UDPE检测方法的建立和应用

目的：建立防止产物污染的UDPE（UDG－Duplex PCR－EIA）技术，用于检测类鼻疽伯克霍尔德氏菌。方法：选择类鼻疽伯克霍尔德氏菌FUR（Ferric Uptake Reg ulator）基因为靶序列，设计两对引物进行PCR扩增，用UDG（尿嘧啶糖基化酶）防止PCR产物污染，并用微孔板杂交－酶联显色检测扩增的PCR产物片段；用不同的模板考察方法的特异性和灵敏度。结果：该检测系统可以特异性地检测类鼻疽伯克霍尔德氏菌；对于纯DNA模板，检测灵敏度可以达到10fg/μl；对于系列稀释菌液提取的模板，灵敏度可达0.1个菌／μl；对于模拟污水标本、模拟组织标本和模拟土壤标本的检测灵敏度分别为10个菌／μl，100个菌／μl和100个菌／μl；检测系统可以防止10^9个PCR产物分子的污染；检测系统可以在37℃下稳定保存7d。结论：本研究建立了稳定的，防止产物污染，灵敏度特异性均较理想的类鼻疽伯克霍尔德氏菌UDPE检测系统。     

鼠疫胶体金法快速诊断试剂盒的研制

目的 研制一种敏感、特异、快速并适用于非专业人员的鼠疫胶体金法快速诊断鼠疫FI抗体的试剂盒。方法 采用柠檬酸三钠还原法制备胶体金颗粒，标记金黄色葡萄球菌A蛋白作为探针，同时用鼠疫多糖抗原致敏硝酸纤维素膜，共同组装成基于免疫层析原理的胶体金法快速诊断盒。结果 间接血凝法检测阳性300份血清标本，胶体金法检测全部为阳性，间接血凝法检测阴性100份血清标本，胶体金法检测全部为阴性，两种方法符合率100％；并且鼠疫胶体金法快速诊断盒不与假结核耶尔森氏菌等相关细菌的免疫血清发生交叉反应。结论 鼠疫胶体金法快速诊断盒具有较好的特异性和敏感性，并且具有更简便快速的优点，适合基层单位现场使用。     

Simple dipstick assay for the detection of Salmonella typhi-specific IgM antibodies and the evolution of the immune response in patients with typhoid fever

Application of a dipstick assay for the detection of Salmonella typhi-specific IgM antibodies on samples collected from S. typhi or S. paratyphi culture-positive patients at the day of admission to the hospital revealed the presence of specific IgM antibodies in 43.5%, 92.9%, and 100% for samples collected 4-6 days, 6-9 days, and > 9 days after the onset of fever, respectively. The mean sensitivity for samples collected an average of 6.6 days after the onset of fever was 65.3%. Culture was positive in 65.9% of the cases with a final clinical diagnosis of typhoid fever. Testing of paired serum samples from culture negative patients with a final clinical diagnosis of typhoid fever resulted in staining of the dipstick in 4.3% of the samples collected at the day of admission to the hospital and in 76.6% of the samples collected one week later, thereby provided strong supporting evidence of typhoid fever by demonstrating seroconversion in a large proportion of the patients. The dipstick assay may thus also be useful for the serodiagnosis of culture-negative patients with clinical signs and symptoms consistent with typhoid fever. The advantages of the dipstick assay are that the result can be obtained on the same day allowing a prompt treatment, that only a small volume of serum is needed, and that no special laboratory equipment is needed to perform the assay. The stability of the reagents of the dipstick and the simplicity of the assay allows its use in places that lack laboratory facilities.     

International multi-centre evaluation of a dipstick assay for human leptospirosis

A dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera was evaluated in 27 laboratories in 23 countries. 873 serum samples from 711 patients including 329 laboratory-confirmed leptospirosis case patients, 239 noncase patients and 69 patients with viral infections causing heamorrhagic fever were tested. Relative to the results of the reference leptospirosis test, the sensitivity of the dipstick assay was 84.5% for serum samples collected during the first 10 days of the disease and 92.1% for serum samples collected 10-30 days after the onset of disease. The specificity was 87.5% and 94.4%, respectively. Similar to viral haemorrhagic fevers, leptospirosis may cause bleeding. A small number of serum samples from patients with haemorrhagic viral infections gave a weak (1 +) stain. All other samples were negative. In conclusion, the dipstick assay is sensitive and specific and reacts well with serum samples from patients infected with a range of leptospiral strains. It is also easy to use and does not require special equipment or refrigeration. Therefore the assay is ideal for use in developing countries and rural settings.     

用重组 rK39抗原试纸条快速诊断内脏利什曼病

[目的 ]评价以恰氏利氏曼原虫类 kinesin基因中编码 39个氨基酸的基因片段 (r K39)为重组抗原 ,用于血清学诊断内脏利什曼病的价值。 [方法 ]在新疆喀什地区对 13例经脾检和骨髓穿刺阳性的内脏利什曼病患者 ,取一滴病人全血或血清滴在 r K39抗原试纸条底部的吸收垫上 ,血清中蛋白随缓冲液向试纸条上部移动 ,其中相应特异抗体可与 r K39抗原带结合 ,而产生阳性条带。同时 ,本文亦用相同阳性血清作了关于 r K 39抗原的 Western印迹分析对照。 [结果 ]EL ISA分析显示病人血清抗体滴度在 10 - 2～ 10 - 4 ,与所见到的 r K39试纸条上的反应强度符合。Western印迹分析亦显示阳性血清可识别 r K39蛋白条带。[结论 ]与传统诊断内脏利什曼病方法相比较 ,r K39试纸条更快速 ,特异 ,灵敏和低损伤性 ,可用于低发病率流行区的内脏利什曼病的诊断和筛选     

Antibody response in typhoid fever in endemic Indonesia and the relevance of serology and culture to diagnosis

Culture and serology were performed on blood and serum samples collected at or shortly after admission from 473 patients presented with suspected clinical typhoid. Clinical symptoms at first presentation including confusion, hepatomegaly, splenomegaly, abdominal pain, anemia, and gastrointestinal bleeding were non-specific as they were observed even more often in non-typhoid patients. Culture confirmed the diagnosis in 65.3% of the patients with typhoid fever as the final diagnosis. The sensitivity (58%) and specificity (98.1%) of a rapid dipstick assay for the detection of S. typhi-specific immunoglobulin M were somewhat lower than those of culture but higher than those of the Widal test. The dipstick assay thus may well be used in the serodiagnosis of typhoid in situation where culture facilities are not available. Combination of test results of dipstick and culture improved sensitivity to 82.5%. In laboratories that perform blood culture the dipstick assay may be used as a rapid screening tests to facilitate a rapid diagnosis. Sensitivity of the dipstick assay strongly increased with duration of illness and was higher for culture positive than for culture negative patients. Duration of illness, and different pathogen and host factors including dose of infection, pathogenicity and antigenicity, and prior antibiotic use are likely to influence the immune response, therefore the result of the dipstick assay. Duration of illness and presence of S. typhi in the blood are major factors that determine severity of disease.     

Detection of filaria-specific IgG4 antibodies and filarial DNA, for the screening of blood spots for Brugia timori

The establishment of simple, sensitive and specific tools for the diagnosis of brugian lymphatic filariasis is a prerequisite for a successful intervention to control the disease. In the simple and rapid Brugia Rapid (BR) test, an immunochromatographic dipstick is used to detect IgG(4) antibodies that are reactive with a recombinant Brugia malayi antigen. When sera from 109 individuals with Brugia microfilaraemias (12 with B. malayi and 97 with B. timori) were investigated using the BR test, all were found positive. In contrast, all of the 150 sera from individuals with Onchocerca volvulus or Mansonella infections investigated were found negative in BR tests. Some unwelcome cross-reactions were observed, however, with sera from individuals infected with Wuchereria bancrofti (three of 12 test-positive) and Dirofilaria (one of nine test-positive). In an attempt to facilitate sample collection and detect any cross-reactions, the BR dipstick was used to screen blood spots, that had been allowed to dry on filter paper, for B. timori microfilariae, before the dipstick-positive samples were tested with a PCR-based assay. Of the 66 individuals so tested, 37 (56%) were found positive by the BR test used on dry blood spots and eight (22%) by the filtration of fresh blood samples. Only nine of the 37 dipstick-positive samples were found PCR-positive. The combined use of BR tests and PCR-based assays, for testing blood spots in areas where brugian filariasis is endemic, appears to be a promising method not only for post-treatment monitoring but also for the certification activities planned within the framework of the Global Programme to Eliminate Lymphatic Filariasis.     

Introduction of a rapid dipstick assay for the detection of Leptospira-specific immunoglobulin m antibodies in the laboratory diagnosis of leptospirosis in a hospital in Makassar, Indonesia

An easy, rapid and robust dipstick assay for detection of leptospira-specific immunoglobulin M (IgM) antibodies was evaluated on 403 patients admitted for hospitalization because of fever. The clinical symptoms and signs of 35 patients were consistent with leptospirosis. The final diagnosis for the remaining patients was as follows: 136 with typhoid fever, 82 with hepatitis, 74 with malaria, 48 with infections of the respiratory tract, and 20 with fever of unknown origin. The clinical diagnosis of leptospirosis was confirmed for 24 (68.6%) patients by the combined results of the microscopic agglutination test (MAT), the reference test for leptospirosis, and of IgM ELISA, a standard laboratory test for the serodiagnosis of leptospirosis. In addition, serum specimens from 8 (2.2%) patients with a final clinical diagnosis other than leptospirosis were found to be positive in MAT and/or IgM ELISA. Compared with the results of MAT and IgM ELISA a sensitivity of 91.6% and specificity of 93.6% was calculated for the dipstick assay. Most of the serum samples from the laboratory confirmed patients gave a moderate to strong staining intensity of the antigen band of the dipstick and were easy to read. The results demonstrate that the dipstick assay is convenient to use and allows the rapid and accurate confirmation of patients with clinical suspicion of leptospirosis in areas where the disease is endemic.     

Prolonged incubation in the two-colour immunofluorescence test increases the prevalence and titres of islet cell antibodies in Type 1 (insulin-dependent) diabetes mellitus

Summary The conventional indirect immunofluorescence test of islet cell antibodies was recently improved by the development of a two-colour immunofluorescence assay using a monoclonal proinsulin antibody to detect islet B cells. The aim of this study was to test whether in this new assay the prevalence and titre of ICA were affected by the time of incubation carried out in the presence of aprotinin (Trasylol) as an inhibitor of proteolysis. The end-point titre of ICA was therefore determined in sera from 70 children aged 0.6 to 15 years with recent onset Type 1 (insulin-dependent) diabetes mellitus, 50 healthy control subjects and 97 non-diabetic siblings of Type 1 diabetic children. In the conventional twocolour assay, ICA was positive in 53/70 (76%) Type 1 diabetic patients, 1/50 control subjects and 2/97 siblings after 30 min incubation. Prolonged incubation for 18 h increased the prevalence of ICA positive samples to 62/70 (89%) in the diabetic patients and to 2/50 in the control subjects, while the prevalence among the siblings was unchanged. Of the ICA positive non-diabetic subjects, one control child has a father with Type 1 diabetes, and one of the siblings subsequently developed Type 1 diabetes. In the diabetic patients the median titre was 1:32 for the 30 min incubation, and it increased to 1:64 for the 18 h incubation (p<0.001). A marked prozone effect was seen; 16% of the samples from the Type 1 diabetic children sera were negative at a 1:2 dilution, but were found positive at higher dilutions. In conclusion, an 18 h incubation increases the end point titres and the prevalence of ICA in the two-colour ICA assay in Type 1 diabetic children of recent onset. The prevalence and levels of ICA among these patients may be larger than hitherto expected.     

不同血型成人胰腺用于ICA检测的比较研究

目的应用免疫组化法(ABC法)检测ICA,比较研究A型、B型、AB型与O型4种不同血型成人胰腺抗原底物对ICA检测的特异性和敏感性的影响。方法取24例1型糖尿病病人的ICA阳性不同JDF单位的血清标本和23例正常人ICA阴性血清标本,以A型、B型、AB型与O型4种不同血型成人胰腺的冰冻切片为抗原底物,应用ABC免疫组化法检测ICA。结果以O型血成人胰腺为对照,应用3例A型和B型血胰腺和2例AB型血胰腺为抗原底物进行ICA检测,24例ICA阳性血清和23例ICA阴性血清检测结果的特异性和敏感性基本一致,未见假阳性或假阴性结果。结论本文研究结果证明A型、B型或AB型血成人胰腺与O型血成人胰腺一样,可用于ICA免疫组化(ABC法)检测,不同血型人胰腺作为抗原底物不影响ICA免疫组化法检测的特异性和敏感性。     

Non-human primate pancreas as a substrate for the detection of islet-cell antibodies in human sera

In an effort to find out if the use of non-human primate pancreas may improve the sensitivity of the islet cell antibody (ICA) test, the sera from patients with type 1 diabetes (insulin-dependent IDDM) and controls were investigated by indirect immunofluorescence (IFL) on both human and baboon substrates. The mean titers of positives were insignificantly higher (1:24.9) on baboon as compared to human tissue (1:22.3). Of 50 sera from IDDM patients positive for ICA on human tissue, 47 were also positive on baboon pancreas. Of 40 ICA-negative IDDM sera two were judged positive on baboon substrate. ICA were positive on human/baboon pancreas in 3/4 out of 50 first-degree relatives of IDDM patients, 2/2 of 50 sera from patients with autoimmune diseases, 0/0 of 50 sera from type 2 diabetics and 2/1 of 100 mixed hospital controls. A disadvantage of baboon pancreas for ICA testing by IFL is the high background fluorescence given by the exocrine pancreas. With baboon tissue, three highly positive results would have been missed with undiluted sera which are usually used in this assay. It is therefore suggested that human pancreas should still be preferentially used for ICA determination, but baboon tissue may be a valuable substitute.