Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA

【Objective】 This study was intend to obtain cathepsin L1(rFgCat L1) specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】 Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened, 1×10⁶ cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method, antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody, and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control, and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】 After immunization, the antibody titers in serum of 4 mice were all more than 10⁴.After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures, the antibodies secreted in the cell supernatant were stable, with titers of 2⁹ and 2¹⁰ respectively, with ascites titers of 10⁷ and 10⁸.Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type, both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies, the antigen titer of the two monoclonal antibodies were comparied, 7G6 was used as the coating antibody, and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.     

绵羊和水牛实验感染大片吸虫后外周血白细胞计数的分析

对绵羊和水牛在感染大片吸虫后外周血白细胞总数计数和白细胞分类计数(嗜酸性细胞、嗜中性细胞、嗜碱性细胞、淋巴细胞和单核细胞)的特性进行研究。结果表明,绵羊对大片吸虫很不易感,水牛对大片吸虫很易感。未感染绵羊和水牛的白细胞总数有较大的差距,分别为5 956(±825)个细胞/mm3血液和12 657(±1 053)个细胞/mm3血液。未感染绵羊和水牛的嗜酸性细胞占白细胞总数的百分比小于5%。淋巴细胞占的比例最大,绵羊为59.5%,水牛达70.8%。感染绵羊和未感染绵羊的白细胞总数、嗜酸性细胞计数在感染过程中总体上存在显著差异,感染后绵羊的嗜酸性细胞的数量迅速升高,在感染后4周(4W P I)达到高峰。而感染水牛和未感染水牛白细胞总数差异不显著,但嗜酸性细胞存在显著差异,感染后水牛的嗜酸性细胞的数量升高缓慢,到8 W P I达到高峰。结果提示嗜酸性细胞在感染绵羊中更加有效地参与了杀灭大片吸虫。     

抗大片吸虫ES抗原单克隆抗体的制备及特异性鉴定

用大片吸虫分泌排泄（ES）抗原免疫BALB/c小鼠，取其脾细胞与SP2/0骨髓瘤细胞融合，经筛选和3次克隆化培养后获得2株分泌抗大片吸虫ES抗原单抗的杂交瘤细胞（F5，F9）。经传代、冻存、复苏培养检测和染色体检查，证实其分泌单抗的性质稳定。通过间接ELISA试验证明，F5和F9单抗能与大片吸虫、肝片吸虫的ES抗原发生特异性反应，而不与它们的虫体抗原反应。SDS-PAGE电泳和Westerm-blot显示，F5McAb能与ES抗原的相对分子质量为26000-28000的蛋白条带反应，而不与其可溶性虫体抗原反应。表明F5McAb有ES抗原特异性，是一株具有潜在诊断价值的McAb。     

片形吸虫分泌排泄抗原和虫体抗原的分析

用牛源肝片吸虫（南京）、牛源大片吸虫（广西）和羊源肝片吸虫（实验感染）制备可溶性虫体抗原（BA）和和分泌排泄抗原（ES），以SDS－PAGE电泳分析比较，结果显示，3株虫体可溶性虫体抗原至少有20条以上的主要蛋白条带，分子量集中于10－90KD之间，它们之间无显著差异，而3株分泌排泄抗原蛋白成分较简单，明显可分的条带不超过5条，分子量集中于10－30KD之间，且均拥有26－28KD的蛋白成分，提示该蛋白应为片形吸虫ES抗原的主要免疫成分。     

大片吸虫组织蛋白酶L全长序列的获取及其生物信息学分析

根据文库载体序列与原组织蛋白酶表达序列标签（EST）设计引物,以大片吸虫cDNA文库为模板,进行Touchdown PCR与梯度PCR相结合的扩增反应,扩增产物克隆人pMD18-T载体。经鉴定后测序,并采用生物信息学技术对序列进行开放阅读框（ORF）、编码氨基酸序列、蛋白质同源性比较、二级、三级结构等分析;结果所获序列全长990bp,编码330个氨基酸,分子量36771.2u,等电点5．44;具有较明显的螺旋、片层和无规卷曲等二级结构,α螺旋占17．3％,β折叠占27．0％,无规卷曲占55．8％;具有2个较明显的跨膜区和2个疏水区,未发现明显的信号肽和糖基化位点;氨基酸序列同源性比对显示其属于半胱氨酸家族;三级结构同源建模预测显示其与组织蛋白酶K（PDB number 2f7dA）有49％的一致性。     

大片吸虫分泌排泄抗原对小鼠肝损伤的研究

为探究大片形吸虫分泌排泄物对小鼠肝脏的影响,将60只雌性昆明小鼠随机分为试验组和对照组各30只。每隔2天注射0.4mg FgESP 200μL,对照组注射PBS 200μL。于首次注射后第10天、4周、7周、14周、18周处死6只小鼠,观察肝脏表观病理变化,制做肝脏HE染色病理切片并测定血清中与肝功能密切相关的ALT、AST、ALP、ALB、GLB等生化指标的变化。肝脏眼观病理变化结果显示,试验组从第4周可见白色点状结节,症状随着时间的延长而加重;肝脏组织病理结果显示,从第2周开始小鼠肝脏出现空泡变性,至第18周出现大量炎性细胞浸润,并伴随轻度肝纤维化;生化指标动态变化结果显示,ALT、AST活性在整个试验进程中出现显著上升(P0.05),ALP活性在第4周~7周明显升高,之后降低(P0.05),ALB和GLB水平在整个试验进程中分别表现为显著下降和显著上升(P0.05),提示肝细胞可能发生变性、坏死导致血清中转氨酶活性升高,蛋白合成能力下降。本研究结果表明大片吸虫分泌排泄抗原(FgESP)可导致小鼠肝损伤,损伤程度随着FgESP的持续注射而加重。     

Development and Application of Sandwich ELISA for Diagnosis of Fascioliasis gigantica in Early Stage

To establish a method for the diagnosis of Fascioliasis gigantica in early stage, five hybridoma cell lines were recovered and used for preparation of monoclonal antibodies.7D2 was used as a capture antibody and 7D1 as detection antibody.Coating antibody dilution was 1:6 400 (0.208 μg/mL), 5% nonfat milk was used as blocking solution and detection antibody dilution was 1:10 000 (0.200 μg/mL).The detection limit of the sandwich ELISA was 1:3 200 (0.156 μg/mL), with good specificity and stability, and there was no cross reaction with other kinds of parasite antigen.The results showed that the method provided the important conditions and theoretical basis for the diagnosis of Fascioliasis gigantica in early stage. This would save unnecessary economic losses to the livestock, and it had clinical application value.     

抗大片吸虫分泌排泄抗原单克隆抗体制备

【目的】制备大片吸虫分泌排泄抗原单克隆抗体,为大片吸虫病的免疫诊断、防治等研究奠定基础。【方法】利用杂交瘤技术,以大片吸虫分泌排泄抗原为免疫原制备单克隆抗体,并用ELISA、硫氰酸盐洗脱法等方法鉴定其生物学特性。【结果】通过间接ELISA筛选及3次亚克隆后,共获得5株大片吸虫分泌排泄抗原单克隆抗体杂交瘤细胞株,分别命名为6D3、6B4、7D2、7D1和7D4。经鉴定,发现5株杂交瘤细胞染色体数为90～110条,均大于亲本细胞,其亚类鉴定分别属于IgG2b、IgM、IgM、IgG1和IgM,其轻链均为κ型;5株单克隆抗体（6D3、6B4、7D2、7D1、7D4）的上清效价分别为1∶400、1∶3200、1∶25600、1∶6400和1∶6400,腹水效价分别为1∶104、1∶104、1∶105、1∶107和1∶106;而表位测定结果表明,5株单克隆抗体中,6D3与7D1、7D2以及7D4与7D1、7D2作用于不同表位,6D3与6B4 可能识别同一表位或重叠表位,或同一表位有空间位阻,7D4与6D3以及6B4与7D4、7D1、7D2可能具有空间位阻;5株单克隆抗体的相对亲和力为：6B4＞7D4＞7D1＞6D3＞7D2;5株杂交瘤细胞株均能稳定地分泌单克隆抗体。【结论】制备获得的分泌排泄抗原单克隆抗体可用于大片吸虫病的诊断和免疫机理等方面的进一步研究。     

Comparative Parasitological and Haematological Changes in Two Breeds of Sheep Infected with Fasciola gigantica

Twelve each of Red Masai and Dorper sheep, aged between 6 and 9 months, were acquired from a Fasciola-free area of eastern Kenya. Each breed was divided into two groups of 6. The sheep in one group of each breed were experimentally infected with 400 viable metacercariae of Fasciola gigantica. The other group of 6 sheep of each breed remained as uninfected controls. The animals were monitored regularly for any evidence of disease. Blood samples taken weekly revealed a general reduction in red cell counts and packed cell volume, which was much faster in the infected Dorper sheep than in the Red Masai. This reduction started from the tenth week after infection and persisted to the end of the experiment 18 weeks post infection (PI). The absolute eosinophil counts rose in all the infected animals, but the values were higher among the Dorper than among the Red Masai. Patency occurred at weeks 12 and 13 PI in the Red Masai and Dorpers, respectively, with the latter shedding significantly more fluke eggs. The worm recovery rates were higher among the Dorpers than among the Red Masai, though not significantly so. On the basis of egg counts and clinicopathology, the Dorper sheep were considered to be more susceptible to F. gigantica infections.     

Bayesian hierarchical modelling to enhance the epidemiological value of abattoir surveys for bovine fasciolosis

Four classes of Bayesian hierarchical models were evaluated using an historical dataset from an abattoir survey for fasciolosis conducted in Victoria, Australia. The purpose of this analysis was to identify areas of high prevalence and to explain these in terms of environmental covariates. The simplest of the Bayesian models, with a single random effect, validated the use of smoothed maps for cartographic display when the sample sizes vary. The model was then extended to partition the random effect into spatially structured and unstructured components, thus allowing for spatial autocorrelation. Rainfall, irrigation, temperature-adjusted rainfall and a remotely sensed surrogate for rainfall, the normalised difference vegetation index (NDVI), were then introduced into the models as explanatory variables. The variable that best explained the observed distribution was irrigation. Associations between prevalence and both rainfall and NDVI that were significant in fixed effects models were shown to be due to spatial confounding. Nevertheless, provided they are used cautiously, confounded variables may be valid predictors for the prevalence of disease.