猪群血液内麻疹血凝抑制抗体样物质的观察研究

目的 了解麻疹血凝抑制抗体样物质在猪群中的分布情况。方法 采用麻疹血凝抑制试验,并用白陶土法去除非特异性凝集素的干扰。结果 检测１９２份猪血标本,麻疹血凝抑制抗体阳性１５８份,阳性率８２．２９％,平均抗体滴度（ＧＭＴ）１：３．５,以１：２￣１：４的低抗体为主（７８．４８％）。结论 在本地猪群血液中所检出的麻疹血凝抑制抗体样物质,并非由于猪感染与麻疹病毒有近缘关系的牛瘟病毒或犬瘟病毒而出现的一种交叉     

检测受染旋毛虫猪血清中循环抗原对诊断和监测本病的研究

用双抗体夹心-ELISA法检测54头感染旋毛虫的猪血清,33头(61.1±6.7％)呈阳性反应。11头受染猪,于感染后不同时间检测CAg,有10头(90.9％)显示阳性,且绝大多数(8头,72.7％)于感染后3天即出现阳性反应,另于感染后6天和9天各有1头阳性。同时对比检测110头正常猪和两种其它寄生虫病猪,仅有两头(1.8％)感染弓形体的猪出现轻度交叉反应。对北京海淀区某屠宰场1987年自外地引进224头猪,进得流行病学调查,有18头(8.0±1.8％)CAg显示阳性反应,其中且有11头猪同时伴有抗体阳性;另检测陕西省商县50头猪,CAg皆为阴性。     

4头猪耶氏菌感染误诊为布氏菌感染

布病与0∶9型耶氏菌的血清学交叉反应,国外早有报告,国内亦有数篇报告。最近我们发现4份猪血清,按布病常规检测方法判定为布病阳性,经过鉴别试验,确认为耶氏菌感染,类似例子不少,特此介绍,以引起重视。 材料和方法 一、标本来源 在莆田调查布病时,使用布病试管凝集试验法检查猪血清,有4份血清抗体滴度达到1∶100～1∶400,按规定应判为布病阳性。 二、检测方法 1.试管凝集抗原为兰州生物制品研究所产品,在有效期内使用。 2.快速酶斑点试验:在前文的基础上,改用0∶3型耶氏菌外膜蛋白为抗原,布氏菌抗原为全菌可溶性抗原。将这两种抗原滴于同一条膜上,自然凉干后封闭。详细方法和判定见之文献。     

滴金免疫测定法在检测日本血吸虫抗体中的应用

滴金免疫测定法(DIGFA)是以胶体金作为标记物的快速斑点免疫结合试验,抗原、抗体通过渗滤在膜上进行反应,阳性结果出现红色斑点。本文用血吸虫虫卵浸出液为抗原,金标记A蛋白为显示剂,建立了检测血吸虫抗体的DIG-FA。以本法检测血吸虫病患者血清81份,阳性卒92.6％;献血者血清50份,特异性为98％;42份产前检查的孕妇血清均为阴性;6例华枝睾吸虫患者未出现交叉反应;6例并殖吸虫患者中4例出现不同程度的阳性反应,其中3例环卵反应亦为阳性,环沉率8～15％。提示本法的敏感性和特异性与环卵反应相近。     

应用BA-Dot-ELISA检测旋毛虫血清抗体的研究

以旋毛虫肌幼虫层析抗原及国产BA试剂,建立了快速检验旋毛虫病的生物素亲和素酶免疫斑点法(BA-Dot-ELISA)。用此法和Dot-ELISA、PPA-ELISA同时检测15份旋毛虫病人血清,阳性率分别为100％、93.3％和93.3％,检测417份非旋毛虫病人血清,均为阴性。用三种方法检测30份实验感染猪阳性血清,BA-Dot-ELISA敏感性分别为PPA-ELISA和Dot-ELISA的7.13倍和2.70倍。本法与血吸虫病、囊虫病、弓形虫感染者无交叉反应。表明本法对旋毛虫有较高的特异性和敏感性,有助于低水平血清抗体旋毛虫病的检测,适于早期诊断,流行病学调查及动物的宰前检查。     

酶联免疫吸附双抗原夹心法检测人畜布氏菌抗体的研究

目的 为人畜布氏菌病血清学诊断、监测及流行病学调查提供实用和简便的一种酶免疫试验技术。方法 在制备高滴度布氏菌抗原辣根过氧化物酶结合物及其冻干制品基础上，应用酶联免疫吸附双抗原夹心法即双抗原夹心酶联免疫吸附试验（DAgS－ELISA）、试管凝集试验（SAT）及虎红平板凝集试验（RBPT）对从山西省布氏菌病疫区收集的布氏菌病患、布氏菌感染羊、牛、猪以及健康人、羊、牛、猪共计290份血清进行对比检测。结果 上述3种试验对健康人、羊、牛、猪共计140份血清检查均为阴性反应，用DAgS-ELISA对布氏菌感染人、羊、牛、猪共计150份血清检查，其阳性率分别为91．4％、74．6％、66．7％及66．7％，从总体而言，检查的特异性及敏感性，以DAgS-ELISA优于SAT及RBPT。结论 双抗原夹心酶联免疫吸附试验检测人畜布氏菌抗体，不仅特异、敏感、简便，而且制备一种布氏菌抗原酶结合物及其冻干制品，可用于人及各种动物布氏菌抗体的检测，值得推广应用。     

检测两种猪源链球菌抗体间接ELISA方法的筛选与应用

目的建立检测马链球菌兽疫亚种（Streptococcus equi subsp zooepidemius，Sez）与猪链球菌2型（Streptococcus suis type2，Ss2）抗体的间接ELISA方法。方法选用Sez与Ss2的全菌（WAC）、超声波抗原（UA）、英膜多糖（capsular polysaccharide，CPS）作为抗原，筛选最优的检测两种抗体间接ELISA方法。结果3种抗原检测60份免疫后的血清表明，Sez与Ss2的CPS抗体阳性率分别为98．3％、96．7％，WAC为91．7％、85％，UA均为88．3％，前者与后两者差异显著（P〈0．05）。检测免疫前50份血清、免疫后156份血清、60份临床采集血样表明，10％猪免疫前呈现Sez与Ss2的CPS抗体阳性；免疫后CPS抗体阳性率均在90％以上，此结果与免疫保护率相一致；苏州与常州两个猪场的血清各30份，CPS抗体阳性率均在6．7％左右。结论CPS-ELISA方法具有较好的稳定性、特异性、灵敏性，此方法在诊断和流行病学调查方面有价值。     

重组多肽酶联免疫吸附法检测抗Scl-70抗体的初步应用

目的制备人Scl-70抗原207～765位氨基酸片段融合蛋白作为自身抗原,探讨ELISA检测抗Scl-70抗体的敏感性和特异性。方法构建编码Scl-70抗原第207至第765位氨基酸片段的重组体,在宿主菌E.coliBL21（DE3）中表达融合蛋白,经Ni^2＋-NTA亲和层析纯化后,免疫印迹法鉴定抗原性,ELISA检测北京协和医院免疫科血清库中系统性硬化（SSc）及部分其他结缔组织病患者血清抗Scl-70抗体。结果重组融合蛋白在宿主菌中获得可溶性表达,免疫印迹法鉴定表明其能与标准抗Scl-70抗体阳性血清反应,而与正常血清、其他抗血清无反应。在36份SSc患者血清中,天然抗原免疫双扩散（DID）法共检出的6份阳性血清用重组多肽ELISA检测有5份呈阳性,30份经天然抗原DID法检测为阴性的血清用重组多肽ELISA检测有3份呈阳性;20份其他结缔组织病患者血清用重组多肽ELISA检测均为阴性。结论重组的207—765位氨基酸片段是Scl-70抗原的主要抗原表位区域,以重组多肽为基质ELISA检测抗Scl-70抗体的敏感性较高,为进一步研究抗体水平与临床病情变化的相关性奠定了基础。     

畜禽衣原体病间接血凝诊断液的研制与应用

用鹦鹉热衣原体B_(11001)株和沙眼衣原体TE_(55)株以及湖北分离的鹦鹉热衣原体CJ_4、BP_(11)和CW_3等菌株制备的十二烷基硫酸钠(SDS)抗原致敏红细胞与以上菌株的63份高免血清和156份实验感染畜禽血清能发生特异性间接血凝交叉反应。对人工感染5～15天的204份畜禽血清的检出率为100％。感染后5～7天抗体转阳,15～20天达到高峰,一般畜禽抗体可持续3个月,猪在14个月仍保持阳性水平。用此法与直接补体结合(DCF)和间接补体结合(ICF)试验对本省19个县(市)的1430份畜禽血清进行检测。IHA的阳性率:绵羊为21.57％;奶牛为9.20％;猪为23.04％;鸭为41.09％;219份鸡血清两法全为阴性。IHA和CF的总阳性率分别为14.13％和12.03％,两法的符合率为35.15％。     

凝胶扩散-酶联免疫吸附试验(DIG-ELISA)检测猪弓形体病抗体

本文介绍一种简单的DIG-ELISA方法作为定量诊断猪弓形体病新方法。在吸附有抗原的塑料平板上,使抗血清在凝胶中扩散并与平板表面抗原结合,再以酶结合物和底物之间的显色反应,间接地显示抗原抗体反应,测得其直径能定量反应特异性抗体的量。对人工感染猪和健康猪的试验取得满意结果后,进而对现场可疑猪检测并与ELEP对比。二法检出率相似,阳性符合率达94％,与猪瘟无交叉反应。     

酶联免疫法定量检测幽门螺杆菌抗体的研究

对73例十二指肠溃疡和胃炎进行血清抗HP抗体定量测定,结果以尿素酶试验和胃粘膜涂片同时阳性作为HP阳性标准,判断血清抗体法检测HP阳性符合率,以同样方法判断血清抗体法阴性符合率.在尿素酶和胃粘膜涂片阳性39例中,抗HP-IgG抗体阳性35例,阳性符合率为89%;尿素酶和胃粘膜涂片阴性34例中,30例阴性,阴性符合率为88%,5例在临界值范围.经卡方检验差别无显著性(P>0.05).抗HP-IgG定量方法的测定比定性方法更准确.定量方法可得出具体数据,可以判断患者感染细菌时间长短以及反映机体对HP免疫力强弱.HP血清学检查可用于观察抗HP疗效及HP普查.本操作每次应设阴阳性对照,用标准品作为定量标准,标本定量应在24h内完成,以免HP-IgG效价降低,若放置低温冰箱,应避免反复冻融.     

Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection

The CD69 molecule, as an early activation marker of lymphocytes, is often used to assess the activation of cellular immunity. However, for pigs, an anti-pig CD69 antibody is not yet available for this purpose after infection or vaccination. In this study, a monoclonal antibody (mAb) against pig CD69 was produced by peptide immunization and hybridoma technique. One mAb (5F12) showed good reactivity with pig CD69 that was expressed in transfected-HEK-293T cells and on mitogen-activated porcine peripheral blood mononuclear cells (PBMCs) by indirect immunofluorescence assay and flow cytometry. This mAb did not cross-react with activated lymphocytes from mouse, bovine, and chicken. Epitope mapping showed that the epitope recognized by this mAb was located at amino acid residues 147–161 of pig CD69. By conjugating with fluorochrome, this mAb was used to detect the early activation of lymphocytes in PRRSV- and ASFV-infected pigs by flow cytometry. The results showed that PRRSV infection induced the dominant activation of CD4 T cells in mediastinal lymph nodes and CD8 T cells in the spleen at 14 days post-infection, in terms of CD69 expression. In an experiment on ASFV infection, we found that ASFV infection resulted in the early activation of NK cells, B cells, and distinct T cell subsets with variable magnitude in PBMCs, spleen, and submandibular lymph nodes. Our study revealed an early event of lymphocyte and T cell activation after PRRSV and ASFV infections and provides an important immunological tool for the in-depth analysis of cellular immune response in pigs after infection or vaccination.     

ELISA对幽门螺杆菌感染血清IgG,IgA,IgM抗体检测的诊断价值

应用酶联免疫吸附试验（ＥＬＩＳＡ）对无选择性作内镜检查的９０例患者血清，分别作抗ＨＰＩｇＧ，ＩｇＡ，ＩｇＭ特异性抗体的检测，同时与胃粘膜活检组织块的镜检，培养，快速尿素酶试验的结果进行比较。ＥＬＩＳＡ检测结果显示血清ＩｇＧ，ＩｇＡ，ＩｇＭ抗体的阳性率分虽为７１．１％（６４／９０），５６．６％（５１／９０），５６．６％（５１／９０），ＩｇＧ检出率均高于检法６５．６％％（５９／９０），尿素酶试验５５．     

Identification of a family of insulin-like growth factor II secreted by cultured rat epithelial-like cell line 18,54-SF: application of a monoclonal antibody

Somatomedin/insulin-like growth factor (IGF)-like polypeptides (designated SMP) were purified from the serum-free conditioned medium of cultured rat epithelial-like cells, 18,54-SF. A monoclonal antibody (MAb) was produced against partially purified SMP. The antibody was immunoglobulin G1 relatively specific for multiplication-stimulating activity III-2 (rat IGF-II), with a Kd value of 5.6 X 10(-9) M. The antibody showed 100% cross-reactivity with human IGF-II and 10% cross-reactivity with human IGF-I, but did not cross-react with insulin. For purification of SMP, therefore, immunoaffinity chromatography on Sepharose coupled with the MAb was used besides a procedure including ion exchange chromatography, gel filtration, and reverse phase HPLC. The purified SMP (at least five polypeptides) each gave a single peak on reverse phase HPLC and appeared as a single band with an apparent mol wt of 5000-8000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major components of SMP (designated HP1-SMP and HP3-SMP), which were purified about 100-fold from conditioned medium, stimulated DNA synthesis in human fibroblasts in culture and sulfation in chick embryonic cartilage in culture. These polypeptides showed almost the same cross-reactivity as multiplication-stimulating activity III-2 on RIA with the MAb. The partial amino acid sequences of HP1- and HP3-SMP were determined, and these polypeptides were identified with rat IGF-II.     

The N34S mutation of SPINK1 (PSTI) is associated with a familial pattern of idiopathic chronic pancreatitis but does not cause the disease

BACKGROUND: Mutations in the PRSS1 gene explain most occurrences of hereditary pancreatitis (HP) but many HP families have no PRSS1 mutation. Recently, an association between the mutation N34S in the pancreatic secretory trypsin inhibitor (SPINK1 or PSTI) gene and idiopathic chronic pancreatitis (ICP) was reported. It is unclear whether the N34S mutation is a cause of pancreatitis per se, whether it modifies the disease, or whether it is a marker of the disease. PATIENTS AND METHODS: A total of 327 individuals from 217 families affected by pancreatitis were tested: 152 from families with HP, 108 from families with ICP, and 67 with alcohol related CP (ACP). Seven patients with ICP had a family history of pancreatitis but no evidence of autosomal dominant disease (f-ICP) compared with 87 patients with true ICP (t-ICP). Two hundred controls were also tested for the N34S mutation. The findings were related to clinical outcome. RESULTS: The N34S mutation was carried by five controls (2.5%; allele frequency 1.25%), 11/87 (13%) t-ICP patients (p=0.0013 v controls), and 6/7 (86%) affected (p<0.0001 v controls) and 1/9 (11%) unaffected f-ICP cases. N34S was found in 4/108 affected HP patients (p=0.724 v controls), in 3/27 (11%) with wild-type and in 1/81 (1%) with mutant PRSS1, and 4/67 ACP patients (all p>0.05 v controls). The presence of the N34S mutation was not associated with early disease onset or disease severity. CONCLUSIONS: The prevalence of the N34S mutation was increased in patients with ICP and was greatest in f-ICP cases. Segregation of the N34S mutation in families with pancreatitis is unexplained and points to a complex association between N34S and another putative pancreatitis related gene.     

免疫组织化学及PCR技术在淋巴结结核病理诊断中的应用价值

目的 探讨免疫组织化学(immunohistochemistry,IHC)及PCR技术在淋巴结结核病理学诊断中的应用价值。方法 收集首都医科大学附属北京胸科医院病理科保存的2012年1月至2013年7月之间的48例手术根治切除淋巴结结核患者(结核组)和21例非淋巴结结核患者(非结核组)的石蜡包埋组织标本,分别应用IHC染色、荧光定量PCR和抗酸染色法对标本进行检测,以临床最后诊断为金标准,比较各方法的检测效能。结果 IHC染色、荧光定量PCR及抗酸染色检测的敏感度分别为52.1%(25/48)、60.4%(29/48)及27.1%(13/48);IHC染色和荧光定量PCR检测敏感度均高于抗酸染色,差异均有统计学意义(χ ²值分别为 6.27、10.84,P值分别为0.012、0.001);而IHC染色与荧光定量PCR比较,敏感度差异无统计学意义(χ ²=0.68,P=0.411)。IHC染色、荧光定量PCR及抗酸染色法检测非结核组结果均为阴性,特异度均为100%(21/21)。IHC染色、荧光定量PCR及抗酸染色法的阴性预测值分别为47.7%(21/44)、52.5%(21/40)、37.5%(21/56);符合率分别为66.7%(46/69)、72.5%(50/69)、49.3%(34/69),IHC染色、荧光定量PCR均优于抗酸染色法。 结论 IHC染色、荧光定量PCR与抗酸染色法相比,可提高阳性检出率,在淋巴结结核的病理学诊断中具有良好应用价值。     

抗骨桥蛋白抗体对沙鼠肝多房棘球蚴组织周围血管生成的影响

目的观察抗骨桥蛋白（osteopontin,OPN）抗体对长爪沙鼠肝多房棘球蚴（俗称泡球蚴）组织周围血管生成的影响。方法 90只长爪沙鼠随机分为3组,即模型对照组（A组）,兔血清对照组（B组）和抗OPN抗体干预组（C组）,所有沙鼠均采用开腹肝穿刺法接种泡球蚴原头节悬混液0.1ml（约含原头节400个）。B组于接种当天注射兔血清0.15ml/鼠,1次/2d,连续7次,之后改为每周1次,直至处死;C组采用相同的方法注射抗OPN抗体（效价1∶32）;A组不作任何处理。分别与感染后第20、60、100、140、180d每组各处死6只沙鼠,留取肝泡球蚴组织,制作组织切片采用苏木素-伊红（H-E）法和免疫组化Envision法观察各组沙鼠肝泡球蚴组织MVD-CD34的表达情况。结果感染泡球蚴沙鼠肝脏中均可见团块状泡球蚴组织,部分播散至腹腔。感染20d时A、B、C组MVD分别为（9.83±3.87）/HP,（9.67±2.94）/HP和（7.50±1.87）/HP,感染60d时分别为（33.67±3.67）/HP,（32.83±6.11）/HP和（24.33±5.61）/HP,感染100d时分别为（44.67±4.92）/HP,（42.20±6.26）/HP和（28.00±8.76）/HP,感染140d时分别为（34.17±3.19）/HP,（31.67±4.97）/HP和（20.50±4.72）/HP;感染180d时分别为（32.33±7.42）/HP,（29.67±3.88）/HP和（13.50±3.21）/HP。其中感染60d及以后各时间点C组与A组和B组相比,微血管数差异有统计学意义（P〈0.05）。结论抗OPN抗体可抑制沙鼠肝泡球蚴组织周围血管生成。     

Prevalence of Helicobacter pylori Infection in Children from Urban and Rural West Virginia

Our objective was to evaluate the prevalencerate of Helicobacter pylori (HP) in children from urbanand rural areas of West Virginia. In all, 1164 bloodsamples were collected from children who attended a local health fair, pediatric clinics, andemergency departments of four different hospitalslocated in urban and rural counties. Socioeconomicstatus was determined in 303 children. Serum HP antibody (IgG) was measured by enzyme immunoassay (EIA).A total of 468 (40%) samples were HP positive. HPacquisition correlated with increasing age, familycrowding, and community location (urban/rural) but not with gender, water source used (city/well), orsocioeconomic status. The prevalence rate of HP in thechildren of West Virginia is higher than any datapreviously reported from the United States. The results correlated with only few socioeconomiccriteria, suggesting that other factors may contributeto the increased prevalence of HP infection in thechildren of West Virginia.     

Hemostatic plug formation in normal and von Willebrand pigs: the effect of the administration of cryoprecipitate and a monoclonal antibody to Willebrand factor

Hemostatic plug (HP) formation was investigated in the ear bleeding time incision in normal and von Willebrand pigs. HP volume was calculated by integrating the areas of serial sections. In normal pigs (n = 11), platelets immediately formed a layer on the surface of the cut channel. Platelet aggregates formed at the ends of transected vessels and gradually enlarged. Finally, all transected vessels were occluded by HP and bleeding stopped. In contrast, large HPs were formed in the incision in von Willebrand's disease (vWD) pigs (n = 4); these HPs did not cover the ends of the transected vessels, which continued to bleed, allowing the formation of large hemostatically ineffective platelet aggregates in the incision. Canals traversed these HPs, and bleeding from the open vessels may have continued through them. After infusion of cryoprecipitate into a vWD pig, the bleeding time shortened, and the morphological findings of the HPs were similar to those of normal pigs. In normal pigs (n = 3) infused with an anti- Willebrand factor monoclonal antibody, which prolonged the bleeding time, a large HP formed in the incision, similar to that observed in the vWD pig. The volume of the normal and vWD HPs increased with time. These in vivo findings suggest that Willebrand factor is involved in the localization of the HP to the damaged vessel and may also play a role in platelet-platelet interaction. A computerized morphometric technique was used for measuring the volume of the hemostatic plugs and the distance of sequential points on the perimeter of the HP from the center of selected bleeding vessels.     

Increase of lung neutrophils in hypersensitivity pneumonitis is associated with lung fibrosis

Hypersensitivity pneumonitis (HP) is characterized by a T-cell-mediated alveolitis, and the putative role of other inflammatory cells in its pathogenesis remains unclear. In this study we determined whether increased quantities of neutrophils were present in HP lungs, and if they were positive for gelatinase B and collagenase-2. Fifteen nonsmoking patients with subacute/chronic active HP were included. Lung samples were analyzed using myeloperoxidase antibody, and neutrophil/total cell ratio was evaluated by digital processing. All HP tissue samples exhibited variable quantities of neutrophils located inside vessels, and in the interstitial and alveolar spaces. Lung neutrophil percentage ranged from 0.7% to 4.8% (2.1 +/- 1.4%). There was a positive correlation between the percentage of lung neutrophils and the percentage of lung fibrosis (r = 0.6, p < 0.02). Tissue neutrophils showed intense immunoreactive collagenase-2 and gelatinase B staining. Additionally, gelatinolytic activities corresponding to progelatinases A and B and their activated forms, were several-fold increased in the bronchoalveolar lavage fluid (BALF) from patients with HP as compared with control subjects. These findings suggest that in HP lungs there is a persistent traffic of neutrophils loaded with gelatinase B and collagenase-2 that may play a role in the lung damage and in the fibrotic response.