捕食量和温度对麦长管蚜在多异瓢虫体内可测定时间的影响

为了方便准确评价麦田捕食性天敌对麦长管蚜 Sitobion avenae (Fabricius) 的控制作用，应用单克隆抗体及间接 ELISA 方法研究了捕食量和温度对麦长管蚜在多异瓢虫 Adonia variegata（Goeze）体内可测定时间的影响。结果表明：在不同的温度和捕食量下，麦长管蚜在多异瓢虫体内的降解中间产物曲线均为单峰型。温度对猎物的可测定时间有显著的影响，随着温度的上升，猎物的可测定时间不断缩短。特别是当温度达到 30℃时，猎物的降解速率迅速上升，其可测定时间只有 1.18 天。捕食量对猎物的可测定时间也有显著影响，随着捕食量的增加，猎物的可测定时间延长。在 25℃的条件下，当捕食量从 1 头蚜虫增加到 3 头蚜虫，可测定时间从 2.81 天延长到 4.25 天。     

七星瓢虫对麦长管蚜,禾谷缢管蚜捕食选择性研究

本文报导了七星瓢虫对两种猎物捕食选择性研究成果,这对以瓢治蚜工作提供了理论依据。主要结果为:1.七星瓢虫对麦长管蚜有明显的正喜好性,对禾谷缢管蚜有明显的负喜好性。2.瓢虫对两种蚜虫的喜好性与麦长管蚜密度呈显著负相关,与禾谷缢管蚜密度呈正相关。3.当两种猎物共存时,瓢虫对每种猎物的功能反应有所变化。两猎物总密度增加,瓢虫对每种猎物的功能反应越来越趋向Hollink Ⅲ型,一种猎物密度恒定,另一种猎物密度变化时,瓢虫对猎物的功能反应趋向Holling Ⅲ型。而且随猎物恒定的密度增加,HoUing Ⅲ型典型性增加。4.麦长管蚜和禾谷缢管蚜密度增加均降低瓢虫的捕食作用率。但是,禾谷缢管蚜所引起捕食作用率更为严重的减少。     

七星瓢虫对两种麦蚜控制作用的模拟研究

应用二次回归旋转组合设计方法研究了七星瓢虫成虫、幼虫与两种麦蚜共存系统中瓢虫对麦长管蚜和禾谷缢管蚜的捕食量模型.结果表明七星瓢虫对两种麦蚜的捕食量随着瓢虫密度的增加而减少,随着该种麦蚜密度的增加而增加,且七星瓢虫无选择性.七星瓢虫不同个体间的干扰作用对其捕食麦长管蚜数量有显著影响,两种麦蚜数交互作用对七星瓢虫捕食禾谷缢管蚜数量影响显著.该模型可用来预测田间蚜虫的变化,指导麦田蚜虫防治.     

麦长管蚜唾液中几种酶的鉴定、活力测定与功能分析

用Parafilm膜夹营养液法,以两种食料介质饲喂麦长管蚜Macrosiphum avenae 3龄若蚜并收集其唾液,对唾液中的酶类进行了鉴定、活力测定和功能分析。结果表明,在20%蔗糖介质提取液中,鉴定有果胶酶、多酚氧化酶和纤维素酶; 在水介质提取液中鉴定有纤维素酶; 两种介质提取液中都未鉴定出过氧化物酶。酶活力测定结果表明, 在20%蔗糖介质提取液中, 每30头蚜虫分泌的果胶酶、多酚氧化酶和纤维素酶的酶活力分别为2.59×10^-3 U/g、7×10^-3 U/g和7.89×10^-3 U/g; 在水介质提取液中,纤维素酶活力为3.68×10^-3 U/g。行为反应试验结果表明,果胶酶处理麦苗的挥发物组分能引起麦长管蚜寄生性天敌燕麦蚜茧蜂Aphidius avenae和捕食性天敌七星瓢虫Coccinella septempunctata 的嗅觉偏好反应,因此,果胶酶在麦长管蚜取食诱导小麦植株的间接防御反应中具有重要作用。     

七星瓢虫幼虫对两种麦蚜的功能反应

七星瓢虫各龄幼虫对麦长管蚜和禾谷缢管蚜的捕食作用均属Holling Ⅱ型反应;其喜食性及发育历期无明显差异;因而两种麦蚜都可互为它的替代猎物而不影响其正常发育。并提出采用α′曲面、T_k曲面以及α′/T_k来衡量天敌对害虫的控制作用;在田间应以3、4龄七星瓢虫幼虫和成虫为主要应用对象;应保留一定蚜量以满足瓢虫发育。     

双抗体夹心ELISA检测HIV-1 gp41抗原方法的建立

目的:建立检测HIV-1gp41抗原的双抗体夹心ELISA,并探讨其临床应用的可行性。方法:用饱和硫酸铵(SAS)纯化抗HIV-1gp41-5单克隆抗体(mAb),用HRP标记后建立双抗体夹心ELISA法,对其灵敏度及特异性进行检测,并用该方法对40份HIV-1阳性血清进行了检测。结果:用mAbE12(5μg/mL)为包被抗体,2H6为酶标记抗体(1∶900)建立了双抗体夹心ELISA法,检测gp41-5多肽的灵敏度是100pg/mL。对HIV-1阳性血清中gp41抗原的检出率为67.5%(27/40)。结论:建立了特异性强、灵敏度良好的检测HIV-1gp41抗原的双抗体夹心ELISA法。     

普通草蛉幼虫对麦二叉蚜和麦长管蚜的捕食功能反应与搜寻效应

[目的]为评价普通草蛉Chrysoperla carnea幼虫对麦二叉蚜Schizaphis graminum和麦长管蚜Sitobion avenae的捕食反应和选择偏好,以明确普通草蛉幼虫对这2种蚜虫的控害能力.[方法]室内设置不同密度的麦二叉蚜和麦长管蚜,统计普通草蛉2龄和3龄幼虫对2种猎物的捕食量,研究普通草蛉幼虫对麦二叉蚜和麦长管蚜的捕食功能反应和搜寻效应.[结果]相同猎物密度下,普通草蛉2龄、3龄幼虫对麦二叉蚜的捕食量均低于麦长管蚜且存在显著性差异,对2种小麦蚜虫的捕食功能反应均拟合Holling Ⅱ功能反应模型和Holling Ⅲ功能反应新模型.3龄幼虫对麦二叉蚜和麦长管蚜的瞬时攻击率分别为1.089和1.106,大于2龄幼虫对猎物的瞬时攻击率,同一龄期草蛉幼虫对麦长管蚜的瞬时攻击率及日最大捕食量大于麦二叉蚜,2龄和3龄幼虫对麦长管蚜的处理时间为0.005 d和0.004 d,均小于对麦二叉蚜的处理时间.普通草蛉幼虫对麦长管蚜的最佳寻找密度高于麦二叉蚜,其中2龄普通草蛉幼虫捕食麦长管蚜的最佳寻找密度最高,为39.200.普通草蛉幼虫对小麦蚜虫的搜寻效应随猎物密度增加而降低,对麦长管蚜的搜寻效应高于麦二叉蚜,麦长管蚜搜寻效应的下降趋势大于麦二叉蚜.[结论]普通草蛉幼虫对麦二叉蚜和麦长管蚜有较大的控害潜能,对于麦长管蚜的取食及控制能力高于麦二叉蚜.     

一种人抗PD-L1单抗的间接ELISA方法的建立

[目的]制备一种人抗PD-L1抗体,建立其酶联免疫吸附分析(ELISA)的检测方法。[方法]将表达抗体重链和轻链的质粒转染到HEK-293细胞制备抗体,并建立ELISA方法对抗体进行检测。[结果]间接ELISA法的最佳抗原包被浓度为0.25μg/mL,抗PD-L1抗体标准品的起始浓度为0.25μg/mL,最适封闭液浓度为2%BSA,最适封闭时间为2 h,二抗的最佳稀释度为1∶4 000。细胞上清中的抗PD-L1抗体样品1的滴度为125,浓度66.66 ng/mL,灵敏度为6.3 ng/mL;样品2的滴度为125,浓度为81.8 ng/mL,灵敏度为5.9 ng/mL。[结论]建立了一种人抗PD-L1单克隆抗体的间接ELISA检测方法,经对样品1和样品2的批内批间重复性试验统计分析,变异系数均10%,该ELISA法适用于该抗体的检测。     

七星瓢虫对麦长管蚜捕食作用及其模拟模型的研究

实验室内16—21℃的温度下,七星瓢虫雌成虫捕食行为集中在8:00—22:00。瓢虫各龄幼虫及雌成虫对麦长管蚜的功能反应均属Holling Ⅱ型。28℃时,瓢虫雌成虫的攻击率最大,处理时间最短。随温度增加,攻击率减小,处理时间增加。假设:猎物种群在无捕食者存在时,呈Logistic曲线增长;捕食者随机搜寻猎物。对猎物的功能反应为Holling Ⅱ型,捕食者个体间存在相互干扰;捕食者种群存在一个最低死亡率K_0,随种群增大,死亡率增加,增加速率与密度成反比;捕食者取食的猎物转化为自身部分的比例为β。 七星瓢虫-麦长管蚜捕食作用系统模拟模型:较好地描述了当麦长管蚜种群增长到某一数量时,放置一头瓢虫雌成虫后蚜虫种群增长过程。本文对模型平衡点作了局部稳定性分析。     

小麦互益素对麦长管蚜及其天敌的影响

研究了在田间使用小麦互益素--水杨酸甲酯和6-甲基-5-庚烯-2-酮对麦长管蚜及其主要天敌--异色瓢虫和燕麦蚜茧蜂的影响.结果表明,小麦互益素虽然没有明显改变麦长管蚜的田间种群变动趋势,但显著降低了麦长管蚜的种群数量.使用小麦互益素能够恶化麦长管蚜的生存环境,使有翅蚜数量明显增加.尽管小麦互益素使天敌物种丰富度有所下降,天敌群落多样性和均匀度指数下降,但燕麦蚜茧蜂、异色瓢虫等优势天敌的种群数量不仅没有减少反而有所增加.因此,田间使用小麦互益素对控制麦长管蚜的危害具有重要作用.     

Development and use of a monoclonal antibody to detect semi-digested proteins of the English grain aphid, Sitobion avenae, in the guts of ladybird beetle predators

A monoclonal antibody (McAb), EGA-4A9, was developed to detect the semi-digested proteins of the English grain aphid, Sitobion avenae (Fabricius) (Hemiptera: Aphididae), in predatory ladybird beetles (species of the genera Adonia , Coccinella , Hippodamia , and Propylea ) using the gut homogenate of Adonia variegata (Goeze) (Coleoptera: Coccinellidae) adults which had fed on S. avenae as immunogen. The McAb was selected by screening hybridoma lines for antibodies that bound with the semi-digested aphid proteins in ladybirds. A double antibody sandwich enzyme-linked immuno-sorbent assay (ELISA) using Clonotyping^TM System/HRP showed that it belonged to the IgG2a isotype. It did not cross-react with any of the 21 arthropod species tested besides the ladybird beetles fed on S. avenae with an indirect ELISA. It could still detect the semi-digested proteins in the gut of a ladybird adult, kept at 25 °C, that had ingested one aphid 6 days before. The extended antigen detection period and the high specificity of the antibody indicated that EGA-4A9 could be used to study interactions between English grain aphids and their ladybird predators in the field. Between 28 and 72% of coccinellids collected from the field were positive for English grain aphid protein by ELISA. The percentage of McAb-positive predatory ladybird beetles was positively correlated with the density of S. avenae in wheat fields.     

降钙素原的原核表达及单克隆抗体制备

目的:高效表达与纯化可溶性重组人PCT蛋白,制备高灵敏度和高特异性的抗人PCT医用诊断单克隆抗体。方法:大肠杆菌表达重组人PCT蛋白后,利用饱和硫酸铵沉淀和亲和层析方法纯化PCT蛋白后,经质谱、Western blot和间接ELISA法进行性质鉴定和分析重组蛋白的表达与免疫反应性;重组蛋白免疫小鼠,经细胞融合及筛选制备抗PCT单克隆抗体(m Ab)。结果:在大肠杆菌中高效表达了人PCT蛋白;重组人PCT蛋白具有良好的免疫反应性与免疫原性;经筛选获得7株抗PCT单克隆抗体细胞株,经ELISA鉴定,筛选抗体可与PCT抗原有良好的特异性反应。结论:利用重组人PCT蛋白免疫制备了抗人PCT单克隆抗体,为进一步研发PCT快速诊断试剂提供了原料。     

Assay sensitivity and differentiation of monoclonal antibody specificity in ELISA with different coating buffers

Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated.     

Monoclonal antibodies reveal changes in predator efficiency with prey spatial pattern

Spatially explicit predator–prey interactions can alter the predatory potential of natural enemies augmented through conservation biological control. To test hypotheses regarding such interactions and predatory efficiency, we used a combination of molecular techniques and mark–release–recapture to study the foraging behaviour of a generalist carabid predator, Poecilus cupreus , in response to spatial patterns of its cereal aphid prey ( Metapolophium dirhodum and Sitobion avenae ). Beetle and aphid numbers were measured across two grids of sampling locations, within which aphid spatial pattern had been manipulated to generate patchy and more homogenous distributions. Aphid consumption was measured by enzyme-linked immunosorbent assays (ELISA) of beetle gut contents, using an aphid-specific monoclonal antibody. Movement and distribution patterns suggest that P. cupreus does not aggregate at, nor instigate prey-taxis within, aphid patches. However, more than two-thirds of the 2169 P. cupreus tested by ELISA had consumed aphids and the proportion of beetles containing aphid proteins was positively related to aphid density. Against expectation, the proportion of predators feeding on aphids was greatest where prey were homogenously distributed, and this was attributed to the loss of partial refuges for prey in aphid patches. The functional value of this type of uniform foraging strategy is ideally suited to early colonization of the crop habitat, when aphid numbers are low, before populations build up and form strong spatial patterns.     

Indirect ELISA with recombinant GP5 for detecting antibodies to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV), which has six structural proteins (GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.     

In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy

Histoplasma capsulatum contains multiple antigens, among them the H antigen and M antigen, which are useful in serologic testing for histoplasmosis. We prepared 7 mouse monoclonal antibodies (5 IgG, 2 IgM) to histoplasmin, and compared these with polyclonal histoplasmin antibodies raised in rabbits and mice. Both monoclonal and polyclonal antibodies were high titered by ELISA. Colloidal gold immune electron microscopy (CGIEM) showed that polyclonal antibodies to histoplasmin or H antigen bound at multiple sites in the cell wall, cytoplasm, and nucleus of Histoplasma yeast cells. In contrast, antibodies to M antigen selectively label the cell membrane and antibodies to alkali soluble cell wall antigen label only the cell wall. Polyclonal antibodies cross reacted extensively with other fungi, both by ELISA and CGIEM. Monoclonal antibodies stained only cytoplasmic epitopes, but also cross reacted with other fungi by electron microscopy. Only periodate treated H antigen elicited polyclonal antibodies which were more specific than those of untreated H antigen or histoplasmin.     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...     

A comparative study on the efficacy of a pest-specific and prey-marking enzyme-linked immunosorbent assay for detection of predation

The efficacy of two different antigen–antibody combinations to detect predation on eggs of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was compared. The first method was an indirect enzyme‐linked immunosorbent assay (ELISA) using monoclonal antibody‐based gut content analysis that detects H. armigera egg protein. The second method was a sandwich ELISA that detects an exotic protein [rabbit immunoglobulin G (IgG)] applied as an external marker to H. armigera eggs. The target predators were the predatory beetles Dicranolaius bellulus (Guerin‐Meneville) (Coleoptera: Melyridae) and Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). Beetles were fed with H. armigera eggs that had been marked with rabbit IgG and then held at various intervals after prey consumption. Each individual beetle was then assayed by both ELISA techniques to identify the prey remains in their guts. The two ELISA methods were further tested on field‐collected predators. Specifically, protein‐marked egg masses were strategically placed in a cotton field. Then, predators from surrounding cotton plants were collected at various time intervals after the marked eggs were exposed and assayed by both ELISAs to detect the frequency of predation on the marked eggs. The rabbit IgG‐specific sandwich ELISA had a higher detection rate than the H. armigera‐specific indirect ELISA under controlled and field conditions for both predator species. Moreover, a greater proportion of field‐collected D. bellulus tested positive for predation than H. variegata. The advantages and disadvantages of using prey‐marking ELISAs instead of pest‐specific ELISA assays are discussed.     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.