胶体金技术在鸡白痢/鸡伤寒检疫和沙门氏菌O_9抗原检测中的应用研究
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摘要
胶体金技术是继同位素、荧光素、酶标记技术之后的一种新型标记技术，它具有快速、敏感、便捷、不需特殊设备、结果判断直观等优点。沙门氏菌是肠杆菌科中一种重要的人兽共患病病原菌，可引起人和动物的多种沙门氏菌病，而含有O_9抗原的沙门氏菌在其中占据了重要的地位。伤寒沙门氏菌和肠炎沙门氏菌可引起人类严重的疾病；在动物疾病中，鸡白痢沙门氏菌、鸡伤寒沙门氏菌引起的鸡伤寒和鸡白痢是鸡的常见多发性传染病，常给养禽业造成巨大的经济损失。本研究将免疫胶体金技术引入沙门氏菌和沙门氏菌病的诊断，建立了测定鸡白痢／鸡伤寒抗体的脂多糖(LPS)抗原夹心斑点免疫金渗滤法和双抗体夹心测定沙门氏菌O_9抗原的胶体金免疫层析法。
1 斑点免疫金渗滤法快速检测鸡白痢／鸡伤寒抗体方法的建立及其初步应用研究
将鸡伤寒沙门氏菌Sg9 LPS抗原包被于渗滤盒检测区(T区)，针对沙门氏菌O_9抗原单克隆抗体3-47-0包被在质控区(C区)，用胶体金标记鸡伤寒沙门氏菌LPS抗原，建立了一种抗原夹心法快速检测鸡白痢、鸡伤寒沙门氏菌抗体的斑点免疫金渗滤法(DIGFA)。阳性结果为C区和T区均出现清晰的红色圆点，阴性结果仅C区出现红色质控圆点。整个检测过程仅需5分钟。对鸡伤寒沙门氏菌感染鸡阳性血清进行DIGFA检测时，检测区和质控区均能出现清晰的红色斑点；对SPF鸡、大肠杆菌、新城疫病毒感染鸡血清进行检测时，仅在质控区出现清晰的红色
扬州大学硕士论文
斑点。DIGFA比两种玻板凝集试验(PAI，)有更高的灵敏度，人工感染鸡试验表明
在感染后第7天即可从犯只鸡中的7只检测到抗体，在时间上早于PAT。722份
现场血清样品的检疫结果表明，DIGFA的阳性检出率稍高于R钉，(阳性率:DIGFA，
13 .3%; PAr，，12.7%;R汀2，12.3%)，阳性样品抗体几何平均滴度DIGFA大于PAr法
(P(0 .05)。这些结果表明DIGFA快速、灵敏、准确，为鸡伤寒和鸡白痢的检疫
提供了一个新的有效手段。
2胶体金免疫层析条快速检测沙门氏菌。，抗原方法的建立及其初步应
用
本研究通过将抗沙门氏菌09抗原的特异性单克隆抗体4一7一7、羊抗鼠IgG，以条带
状分别包被于硝酸纤维膜的检测区(T)和质控区(C)，用胶体金标记另一株单抗
3一47一0，并将其吸附于金标垫上，成功建立子决速、特异、灵敏的检测沙门氏菌09
抗原的胶体金免疫层析试纸条(GICA)。该试纸条可检测含有场抗原的沙门氏菌，
而不与其它沙门氏菌、肠杆菌科其他细菌和常见革兰氏阳性细菌反应。对鸡伤寒
沙门氏菌599 LPS的最小检出量为0.15n岁条，对599菌液的最小检出量为ZX
1护cFu/条。蛋品的人工污染试验显示，经增菌后免疫层析试纸条可检出1 cFu的
肠炎沙门氏菌，在大肠杆菌与沙门氏菌比例为1000:1仍不干扰试纸条的检测。免
疫层析试纸条可直接对鸡白痢发病鸡进行检测，对于细菌含量较少的带菌鸡或其
它待测样品，只需经过选择性增菌后，即可进行瞬时检测。共对122只鸡进行了检
测，试纸条鸡白痢沙门氏菌检出率为27.9%，与经典细菌分离鉴定法完全符合。市
场采集的鸡蛋样品中试纸条沙门氏菌09抗原的检出率为1 .89%，与经典细菌分离鉴
定法相比敏感度为100%，特异性为99.4%。上述结果表明，检测沙门氏菌场抗原
的GICA法在医学和兽医学重要沙门氏菌病诊断方面有着潜在的广阔前景。
Colloidal gold technique is a novel labeling technique after isotope, fluorescin and enzyme labeling, which has the merits such as easy, rapid, sensitive and ready-to-read. Salmonella are important pathogenic agents for both human beings and animals, from which Salmonella spp. harboring O9 antigen play a big role. Salmonella typhi and Salmonella enteritidis can cause life-threatening typhoid and/or food poisoning in human beings, respectively; in animals, fowl typhoid and pullorum disease are caused by Salmonella pullorum/gallinarum, which usually result in great economic loss. In the present study, colloidal gold technique was used in the diagnosis of salmonellosis and two methods were established: dot immuno-gold filtration assay (DIGFA) to detect the antibody to Salmonella pullorumlgallinarum and gold immunochromatographic assay(GICA) to detect O9 antigen of Salmonellae.
1 Dot immuno-gold filtration assay to detect the antibodies against
Salmonella pullorum lgallinarum
A dot immuno-gold filtration assay (DIGFA) to detect antibodies against Salmonella pullorum/gallinarum was established by using lipopolysaccharide (LPS) as both the coated antigen and the labeling antigen. LPS from Salmonella gallinarum reference strain Sg9 was dotted onto the nitrocellulose membrane to form test region (T-region) and monoclonal antibody 3-47-0 against O9 antigen of Salomenlla was coated on the control region(C-region). Sg9 LPS was also labeled with colloidal gold. If the serum sample contains antibodies against LPS of Salmonella pullorum/gallinarum, red dot will appear in the T-region; if no specific antibody exist, there is no red dot in the T-region. As a control, a red dot will appear in both positive and negative samples. As expected, the sera from chickens infected with Salmonella gallinarum was positive by DIGFA, while sera from specific-pathogen-free (SPF) chickens or chickens infected with E.coli,
Newcastle Disease Virus were negative. DIGFA was more sensitive than two commercially available plate agglutination test (PAT) reagents, as shown by positive sample dilution experiment and artificial infection experiment. DIGFA could detect antibodies specific for Salmonella gallinarum LPS in the seventh day in the 7 out of 32 infected chickens, while PAT needed a longer time to show the positive results. 722 samples were detected by DIGFA and two PAT reagents, the results showed DIGFA could detect a slightly more positive samples (positive rate: DIGFA,13.3%; PAT1, 12.7%; PAT2 12.3%), which was verified by bacteria isolation experiment. DIGFA was more sensitive than the two PAT reagents as demonstrated by the result of the positive serum antibody arithmetical average titer. In summary, DIGFA is rapid, sensitive, specific, which offers a new method to screen fowl pullorum and typhoid disease in chickens.
2 Gold immunochromatographic assay to detect Salmonellae bearing
O9 antigen
A gold immunochromatographic assay (GICA) to detect Salmonellae O9 antigen has been developed. The system employed two different monoclonal antibodies that bound distinct epitopes of Salmonellae O9 antigen: one antibody 4-7-7 immobilized on the surface of a nitrocellulose membrane acting as capture antibody, and the other antibody 3-47-0 labeled with colloidal gold , which was placed in the dry state at a predetermined site on a glass-fiber membrane. The strip could detect Salmonellae containing O9 antigen, but couldn't react with other Salmonellae,other enterobacteria, and Gram-positive bacteria. The detection limit for this strip is 0.l5ng and 2 l0 CFU for Salmonella gallinarum Sg9 LPS and Sg9 strain, respectively. The strip could detect 1CFU Salmonella enteritidis in artificially contaminated eggs after enrichment and E. coli didn't interfere with the detection even at a ratio of 1000:1 with Salmonella enteritidis. In Salmonella gallinarum heavily infected chickens, Salmonellae Og antigen could be directl
y determined in liver or spleen by GICA, while mildly infected chickens, carriers or other samples, could be easily subjected to detection afte
1 斑点免疫金渗滤法快速检测鸡白痢／鸡伤寒抗体方法的建立及其初步应用研究
将鸡伤寒沙门氏菌Sg9 LPS抗原包被于渗滤盒检测区(T区)，针对沙门氏菌O_9抗原单克隆抗体3-47-0包被在质控区(C区)，用胶体金标记鸡伤寒沙门氏菌LPS抗原，建立了一种抗原夹心法快速检测鸡白痢、鸡伤寒沙门氏菌抗体的斑点免疫金渗滤法(DIGFA)。阳性结果为C区和T区均出现清晰的红色圆点，阴性结果仅C区出现红色质控圆点。整个检测过程仅需5分钟。对鸡伤寒沙门氏菌感染鸡阳性血清进行DIGFA检测时，检测区和质控区均能出现清晰的红色斑点；对SPF鸡、大肠杆菌、新城疫病毒感染鸡血清进行检测时，仅在质控区出现清晰的红色
扬州大学硕士论文
斑点。DIGFA比两种玻板凝集试验(PAI，)有更高的灵敏度，人工感染鸡试验表明
在感染后第7天即可从犯只鸡中的7只检测到抗体，在时间上早于PAT。722份
现场血清样品的检疫结果表明，DIGFA的阳性检出率稍高于R钉，(阳性率:DIGFA，
13 .3%; PAr，，12.7%;R汀2，12.3%)，阳性样品抗体几何平均滴度DIGFA大于PAr法
(P(0 .05)。这些结果表明DIGFA快速、灵敏、准确，为鸡伤寒和鸡白痢的检疫
提供了一个新的有效手段。
2胶体金免疫层析条快速检测沙门氏菌。，抗原方法的建立及其初步应
用
本研究通过将抗沙门氏菌09抗原的特异性单克隆抗体4一7一7、羊抗鼠IgG，以条带
状分别包被于硝酸纤维膜的检测区(T)和质控区(C)，用胶体金标记另一株单抗
3一47一0，并将其吸附于金标垫上，成功建立子决速、特异、灵敏的检测沙门氏菌09
抗原的胶体金免疫层析试纸条(GICA)。该试纸条可检测含有场抗原的沙门氏菌，
而不与其它沙门氏菌、肠杆菌科其他细菌和常见革兰氏阳性细菌反应。对鸡伤寒
沙门氏菌599 LPS的最小检出量为0.15n岁条，对599菌液的最小检出量为ZX
1护cFu/条。蛋品的人工污染试验显示，经增菌后免疫层析试纸条可检出1 cFu的
肠炎沙门氏菌，在大肠杆菌与沙门氏菌比例为1000:1仍不干扰试纸条的检测。免
疫层析试纸条可直接对鸡白痢发病鸡进行检测，对于细菌含量较少的带菌鸡或其
它待测样品，只需经过选择性增菌后，即可进行瞬时检测。共对122只鸡进行了检
测，试纸条鸡白痢沙门氏菌检出率为27.9%，与经典细菌分离鉴定法完全符合。市
场采集的鸡蛋样品中试纸条沙门氏菌09抗原的检出率为1 .89%，与经典细菌分离鉴
定法相比敏感度为100%，特异性为99.4%。上述结果表明，检测沙门氏菌场抗原
的GICA法在医学和兽医学重要沙门氏菌病诊断方面有着潜在的广阔前景。
Colloidal gold technique is a novel labeling technique after isotope, fluorescin and enzyme labeling, which has the merits such as easy, rapid, sensitive and ready-to-read. Salmonella are important pathogenic agents for both human beings and animals, from which Salmonella spp. harboring O9 antigen play a big role. Salmonella typhi and Salmonella enteritidis can cause life-threatening typhoid and/or food poisoning in human beings, respectively; in animals, fowl typhoid and pullorum disease are caused by Salmonella pullorum/gallinarum, which usually result in great economic loss. In the present study, colloidal gold technique was used in the diagnosis of salmonellosis and two methods were established: dot immuno-gold filtration assay (DIGFA) to detect the antibody to Salmonella pullorumlgallinarum and gold immunochromatographic assay(GICA) to detect O9 antigen of Salmonellae.
1 Dot immuno-gold filtration assay to detect the antibodies against
Salmonella pullorum lgallinarum
A dot immuno-gold filtration assay (DIGFA) to detect antibodies against Salmonella pullorum/gallinarum was established by using lipopolysaccharide (LPS) as both the coated antigen and the labeling antigen. LPS from Salmonella gallinarum reference strain Sg9 was dotted onto the nitrocellulose membrane to form test region (T-region) and monoclonal antibody 3-47-0 against O9 antigen of Salomenlla was coated on the control region(C-region). Sg9 LPS was also labeled with colloidal gold. If the serum sample contains antibodies against LPS of Salmonella pullorum/gallinarum, red dot will appear in the T-region; if no specific antibody exist, there is no red dot in the T-region. As a control, a red dot will appear in both positive and negative samples. As expected, the sera from chickens infected with Salmonella gallinarum was positive by DIGFA, while sera from specific-pathogen-free (SPF) chickens or chickens infected with E.coli,
Newcastle Disease Virus were negative. DIGFA was more sensitive than two commercially available plate agglutination test (PAT) reagents, as shown by positive sample dilution experiment and artificial infection experiment. DIGFA could detect antibodies specific for Salmonella gallinarum LPS in the seventh day in the 7 out of 32 infected chickens, while PAT needed a longer time to show the positive results. 722 samples were detected by DIGFA and two PAT reagents, the results showed DIGFA could detect a slightly more positive samples (positive rate: DIGFA,13.3%; PAT1, 12.7%; PAT2 12.3%), which was verified by bacteria isolation experiment. DIGFA was more sensitive than the two PAT reagents as demonstrated by the result of the positive serum antibody arithmetical average titer. In summary, DIGFA is rapid, sensitive, specific, which offers a new method to screen fowl pullorum and typhoid disease in chickens.
2 Gold immunochromatographic assay to detect Salmonellae bearing
O9 antigen
A gold immunochromatographic assay (GICA) to detect Salmonellae O9 antigen has been developed. The system employed two different monoclonal antibodies that bound distinct epitopes of Salmonellae O9 antigen: one antibody 4-7-7 immobilized on the surface of a nitrocellulose membrane acting as capture antibody, and the other antibody 3-47-0 labeled with colloidal gold , which was placed in the dry state at a predetermined site on a glass-fiber membrane. The strip could detect Salmonellae containing O9 antigen, but couldn't react with other Salmonellae,other enterobacteria, and Gram-positive bacteria. The detection limit for this strip is 0.l5ng and 2 l0 CFU for Salmonella gallinarum Sg9 LPS and Sg9 strain, respectively. The strip could detect 1CFU Salmonella enteritidis in artificially contaminated eggs after enrichment and E. coli didn't interfere with the detection even at a ratio of 1000:1 with Salmonella enteritidis. In Salmonella gallinarum heavily infected chickens, Salmonellae Og antigen could be directl
y determined in liver or spleen by GICA, while mildly infected chickens, carriers or other samples, could be easily subjected to detection afte
引文
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2. Roth J, Binder. Ultrastruetural Localization of Intracellular Antigens by the Use of Protein A-gold Complex. Histochem Cytoehem. 1978,26: 1074～1081.
3．王文勇，李玉松，赵一岭等．一种快速、稳定制备胶体金的新方法．细胞与分子免疫学杂志，1997，增刊1(13)：18～19．
4．陶义训．固相膜免疫测定．上海医学检验杂志，2000，15(5)：258～260．
5. Spielberg F, Kabeya CM, Ryder RW, et al. Field testing and comparative evaluation of rapid visually read screening assays for antibody to human immunodeficiency virus. Lancet, 1989,1: 580.
6．郑葵阳，杜文平，刘宜升等．快速斑点免疫金染色法检测日本血吸虫病人血清抗体的初步研究．中国现代医学杂志，2002，24(12)：71～72．
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