A3α-肽聚糖对凡纳滨对虾生长、免疫机能和抗病毒感染的影响

通过实验考察了A3α-肽聚糖（PG）制剂对养成期凡纳滨对虾的生长、免疫机能及抗白斑综合征病毒（WSSV）感染能力的影响。设立了连续投喂、间隔投喂和浸浴等6个实验组,分别在30d、60d和90d时,测定对虾的体长、体重、免疫机能和对虾的抗WSSV感染力。结果表明：30d时,连续投喂组对虾的体长和浸浴组及0．05％、0．1％PG连续投喂组对虾的体重较对照组有显著增长（P〈0．05）;0．05％、0．1％PG连续投喂组对虾体内的PO活性较对照组有显著提高（P〈0．05）;60d时,浸浴组及0．05％、0．1％PG连续投喂组对虾的体长、体重较对照组均有极显著增长（P〈0．01）,浸浴组、0．1％PG连续投喂组对虾血细胞吞噬活性显著增强（P〈0．05）,浸浴组及0．05％、0．1％PG连续投喂组对虾血浆上清液PO活性均有显著提高（P〈0．05）;90d时,浸浴组对虾的体长、体重较对照组均有极显著增长（P〈0．01）,间隔投喂组对虾血细胞吞噬活性显著增强（P〈0．05）,间隔投喂组血浆中的PO活性显著增高（P〈0．05）。各期感染实验均证明PG能增强对虾对WSSV的抗感染能力．     

鳗弧菌金属蛋白酶单克隆抗体的制备及鉴定

鳗弧菌M3胞外产物经纯化得到分子量为36.5kDa的金属蛋白酶，浓缩后作为抗原免疫BALB/C小鼠，经细胞融合及筛选。得到三株稳定分泌抗金属蛋白酶的杂交瘤细胞株。分别命名为P2B1，P4A3和P4B1。亚型鉴定结果表明，三株细胞株产生的单抗均为IgG3型。蛋白质印迹（Wes tern Blot)分析显示，所得到的单抗只与分子量为36.5kDa的金属蛋白酶反应，具有较高特异性。三株单抗均能有效抑制蛋白酶活性。     

血细胞分析仪使用中的注意事项

随着特殊溶血剂的应用。能将白细胞分为2～3分群。即淋巴细胞、粒细胞和中间细胞群。这样。细胞计数器的检测项目可多达18项，甚至在实现白细胞五分类后，细胞分析的参数多达20多项，由以前简单的细胞计数器成为名副其实的血细胞分析仪（hematology analyzer）。     

不同对虾种间共用微卫星DNA引物的研究

采用已发表的斑节对虾的30对微卫星引物,对部分微卫星引物在中国对虾、凡纳对虾、日本对虾中的通用性进行了研究。通过所建立的降温PCR(Touchdown PCR)方法对3种对虾的DNA进行PCR扩增,筛选出3对引物在3种对虾中均能产生清晰谱带。根据上述结果适当调整PCR反应体系中各试剂的量及反应条件,达到了理想的结果。利用这3对引物对3种对虾进行遗传标记分析,结果显示,PCR扩增产物条带清晰,特异性强,每对引物在3种对虾间扩增出的特异性片段大小几乎一致。研究表明,3种对虾基因组间存在一定程度的相似性,引物W15-16、W21-22和W45-46可直接应用于中国对虾、凡纳对虾、日本对虾、斑节对虾分子标记的基因组比较作图,也为图谱克隆法提供了新的思路。     

中国对虾组织细胞中酚氧化酶活力的研究

运用酶细胞化学技术，显示中国对虾（Fenneropenaeus chinensis）淋巴器、皮肤、鳃、肝胰腺、中肠育囊和中肠这几种组织细胞中的酚氧化酶活性，在透射电镜下观察各组织中细胞内酚氧化酶的活性部位；观察在受病毒感染的细胞内酚氧化酶对病毒的作用。结果表明，在健康对虾的组织细胞中，基本不表现酚氧化酶活性，而在受病毒感染的对虾中，不同的组织表现不同程度的酚氧化酶活性。其中，尤以淋巴器和皮肤的细胞中酚氧化酶活性最强，在淋巴器和皮肤这两个组织的细胞中，都可看到酚氧化酶对病毒结构的破坏作用。中国对虾淋巴器具有对血淋巴中有害异物进行过滤的作用，酚氧化酶在这一过程中发挥了重要作用。     

中国明对虾(Fenneropenaeus chinensis)和凡纳滨对虾 (Litopenaeus.vannamei )血清酚氧化物酶活性的研究

研究了中国明对虾和凡纳滨对虾血清酚氧化酶（PO）的生理功能和活性影响。结果表明,中国明对虾和凡纳滨对虾血清中都存在PO,主要以酚氧化酶原（proPO)的形式存在,并且都可以被可以被胰蛋白酶、SDS和酵母聚糖激活。抗凝剂SSS（Shrimp Salt Solution)会影响中国明对虾和凡纳滨对虾血清中的PO活性引起的PO活性的降低。正常状态下中国对虾血清中PO活性较凡纳滨对虾血清PO活性低。中国明对虾血清PO最适温度为50℃,最适pH为8．5,而凡纳滨对虾PO最适温度为45℃,最适pH为7．5。     

重金属汞单克隆抗体的制备及鉴定

通过双功能螯合剂(异硫氰基.苯甲基-乙二胺四乙酸,ITCBE)使汞离子与载体蛋白(匙孔血蓝蛋白,KLH)偶联,获得了免疫抗原KLH-ITCBE-Hg(Ⅱ).以该抗原免疫BALB/C小鼠,取免疫小鼠的脾细胞与骨髓瘤细胞融合,杂交瘤细胞用EIJSA方法筛选及有限稀释法亚克隆化,获得了5株可稳定分泌抗汞单克隆抗体的杂交瘤细胞株(E/H9、E/B2、1/F11、1/H7和B/B11).杂交瘤细胞染色体数目在97-104之间,所分泌的抗体均为IgM、k型,其中杂交瘤细胞株E/H9、1/F11和B/B11细胞培养上清液效价分别达1:640、1:320、1:160,腹水效价分别达1:6400、1:3200、1:6400.B/B11的亲和常数为2.981×108L/mol,特异性试验表明:B/B11与其它金属离子的交叉反应率均小于2%,具有较高的特异性.高亲合力和高特异性的抗汞单克隆抗体在该研究中的成功制备,为建立农产品和环境中重金属汞的快速免疫检测方法奠定了基础.     

中国明对虾乙酰基专一性凝集素分离纯化方法的研究

为研究凝集素在中国明对虾天然免疫中的作用,利用3种不同物质--N-乙酰葡萄糖胺(GlcNAc)、胎球蛋白(Fetuin)和小牛黏液素(BSM)为配基,分别与3种柱材料结合进行亲和层析,从中国明对虾血淋巴中纯化凝集素.这3种亲和层析均得到了一种相同的凝集素FCL-1,经SDS-PAGE证明FCL-1的分子量为168kDa.利用新鲜小鼠血细胞进行分离纯化同样获得了这种凝集素.FCL-1与乙酰基糖类和唾液酸类糖蛋白均具有较强的结合特性.     

栉孔扇贝血细胞的吞噬作用及其扫描电镜研究

用透射电镜和扫描电镜技术对栉孔扇贝血细胞的吞噬作用和表面结构进行了研究,结果表明,栉孔扇贝血细胞对大肠杆菌和金黄色葡萄球菌均有很强的吞噬能力,注射细菌15h后,吞噬率分别达25％和27％,小颗粒（嗜中性颗粒）细胞吞噬能力最强,中颗粒（嗜碱性颗粒）细胞吞噬能力较弱,大颗粒（嗜酸性颗粒）细胞无吞噬能力,无颗粒细胞中只有少量较大的细胞具有吞噬能力。吞噬体不断与小空泡（初级溶酶体）融和形成大的吞噬体,并在其内将细菌逐步消化降解,吞噬体中的细菌也可被分散包围成多个小吞噬体后分别被降解。扫描电镜观察栉孔扇贝血细胞的圆形、椭圆形和梭形三种形状,多数血细胞容量变形形成伪足,可形成大量长的纤维状伪足的血细胞具有凝血的功能。     

AFLP分子标记构建中国对虾遗传连锁图谱的初步研究

利用AFLP分子标记结合“拟测交”策略,以中国对虾的单对杂交亲本及其F1代为作图群体,应用MAPMAKER EXP／3．0软件,分别构建了中国对虾雌、雄的遗传连锁图谱。在中国对虾的雄性连锁图谱中,74个标记组成了5个连锁群(遗传标记数量大于3)、7个三联体、13个连锁对,总图距为951．5eM,最大连锁群的长度为206．2eM,最短的为7．4eM,标记间的平均距离为12．8eM;雌性连锁图谱中,由66个标记组成的5个连锁群,4个三联体,13个连锁对,总图距为712．7eM,连锁群的长度介于7．7eM～128．2eM之间,标记间的平均距离为10．7eM。估算的中国对虾基因组总长度为2000eM,所构建的连锁图谱分别覆盖了基因组长度的47．5％和35．6％。中国对虾遗传连锁图谱的构建为分子标记辅助育种、比较基因组作图以及基因定位与克隆创造了条件,并对今后其他海洋生物遗传图谱的构建具有理论价值和实践意义。     

Development of direct ELISA for the determination of 4-nonylphenol and octylphenol

Development of direct competitive enzyme-linked immunoadsorbent assays (ELISAs) based on polyclonal and monoclonal antibodies raised against 4-n-alkylphenol hapten mimics is described. A strong tendency to recognize 4-nonylphenol (NP) and 4-octylphenol (OP) as a total analyte amount was indicated by cross-reactivity pattern established for two polyclonal antibodies. These antibodies were employed for development of class-selective assays exhibiting IC(50) values around 40 microg.L(-1) for technical 4-NP. Specificity of the monoclonal antibody 4H6 and additional two polyclonal antibodies allowed sensitive detection of linear long-chain forms of 4-n-alkylphenols (4-n-AP). The assays incorporating these antibodies offer a potential for detecting the minor fraction of NP/OP isomer spectrum having IC(50) = 11.5 microg.L(-1) for 4-n-NP. No cross-reactivity interference was indicated for linear alkylbenzene sulfonates and phenolic compounds. To interpret the measured data in terms of analytical equivalents, a reliable relationship between the assay responses and AP content of contaminated samples should be verified and validated.     

Intact free prostate-specific antigen and free and total human glandular kallikrein 2. Elimination of assay interference by enzymatic digestion of antibodies to F(ab')2 fragments

Various blood constituents can interfere with immunoassays, usually by binding the Fc portion of antibodies. Our previously developed assays for intact free prostate-specific antigen (PSA), free human kallikrein 2 (hK2), and total hK2 frequently yielded falsely high results despite including an excess of scavenger antibodies. We investigated whether this interference could be eliminated by replacing monoclonal capture or tracer antibodies with F(ab')2 or recombinant Fab fragments. Female heparin plasma samples (n = 1092), which should have negligible PSA and hK2, and male samples (n = 957) were analyzed to identify samples manifesting interference, which then were used to optimize protocols for the immunoassays. We compared original assays (monoclonal antibodies) versus optimized assays (F(ab')2 fragments: denatured mouse IgG added as scavenger) using another set of EDTA plasma (n = 113), heparin plasma (n = 160), and serum samples (n = 171). With the original assays, the frequency of falsely elevated hK2 and intact free PSA was 15 and 13%, respectively. The optimized assays eliminated 70-85% of these falsely elevated results and importantly reduced the magnitude in the remainder. F(ab')2 fragmentation was the most important factor in reducing interference. The optimized intact free PSA, free hK2, and total hK2 assays manifested high accuracy close to the lower limit of detection.     

Multiple-analyte fluoroimmunoassay using an integrated optical waveguide sensor.

A silicon oxynitride integrated optical waveguide was used to evanescently excite fluorescence from a multianalyte sensor surface in a rapid, sandwich immunoassay format. Multiple analyte immunoassay (MAIA) results for two sets of three different analytes, one employing polyclonal and the other monoclonal capture antibodies, were compared with results for identical analytes performed in a single-analyte immunoassay (SAIA) format. The MAIA protocol was applied in both phosphate-buffered saline and simulated serum solutions. Point-to-point correlation values between the MAIA and SAIA results varied widely for the polyclonal antibodies (R2 = 0.42-0.98) and were acceptable for the monoclonal antibodies (R2 = 0.93-0.99). Differences in calculated receptor affinities were also evident with polyclonal antibodies, but not so with monoclonal antibodies. Polyclonal antibody capture layers tended to demonstrate departure from ideal receptor-ligand binding while monoclonal antibodies generally displayed monovalent binding. A third set of three antibodies, specific for three cardiac proteins routinely used to categorize myocardial infarction, were also evaluated with the two assay protocols. MAIA responses, over clinically significant ranges for creatin kinase MB, cardiac troponin I, and myoglobin agreed well with responses generated with SAIA protocols (R2 = 0.97-0.99).     

Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) Hemocytes

Microgravity induces alterations in the functioning of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux-B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reactive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1g operation mode of the clinostat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this purpose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simulated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation) exposed to simulated microgravity (μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels measured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity during parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the centrifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground-based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.     

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.     

Development of xMAP Assay for Detection of Six Protein Toxins

xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2- to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3-200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.