白斑综合证病毒胶体金免疫层析试剂板的研制

将胶体金免疫层析技术应用于对虾白斑综合征的诊断,研制了一种检测对虾中白斑综合征病毒的胶体金免疫层析试剂板.将胶体金标记的抗白斑综合征病毒单克隆抗体包被在金标垫上,将抗白斑综合征病毒多克隆抗体和羊抗鼠IgG分别包被在硝酸纤维素膜的检测线和质控线上.通过双抗体夹心法检测样品中自斑综合征病毒含量.结果表明,该试剂板对白斑综合征病毒的最低检出限为1.0 mg/kg,特异性好,稳定性高,整个检测所需时间为8～10 min,适合现场快速检测.     

白斑综合征病毒胶体金免疫层析试剂板的研制

将胶体金免疫层析技术应用于对虾白斑综合征的诊断,研制了一种检测对虾中白斑综合征病毒的胶体金免疫层析试剂板。将胶体金标记的抗白斑综合征病毒单克隆抗体包被在金标垫上,将抗白斑综合征病毒多克隆抗体和羊抗鼠IgG分别包被在硝酸纤维素膜的检测线和质控线上。通过双抗体夹心法检测样品中白斑综合征病毒含量。结果表明,该试剂板对白斑综合征病毒的最低检出限为1.0 mg/kg,特异性好,稳定性高,整个检测所需时间为8~10 min,适合现场快速检测。     

哈维氏弧菌胶体金免疫层析试纸条的制备

用18~20nm胶体金标记抗哈维氏弧菌Vibrio harveyi抗体并制备金标垫,分别将羊抗兔Ig G和纯化后的抗哈维氏弧菌抗体包被于NC膜上作为质控线和检测线,组装制作了哈维氏弧菌胶体金免疫层析试纸条,优化了该试纸条的制作条件,测试了其性能。结果表明:所制备的哈维氏弧菌免疫层析试纸条具有较好的特异性,与费尼斯弧菌Vibrio furnissii、维氏气单胞菌Aeromonas veronii、美人鱼发光杆菌Photobacterium damselae、类志贺邻单胞菌Plesiomonas Shigelloides、鱼肠道弧菌Vibrio ichthyoenteri、鳗利斯顿氏菌Listonella anguillarum、迟缓爱德华氏菌Edwardsiella tarda、海豚链球菌Streptococcus iniae、嗜水气单胞菌Aeromonas Hydrophila等9种常见水产病原菌无明显交叉反应,检测灵敏度为3×105CFU/m L,5~10min即可得出检测结果,具有快速、简便、特异性强和适应基层推广应用等优点,可用于养殖鱼类病原哈维氏弧菌的现场检测。     

鱼肠道弧菌胶体金快速检测试纸研制

鱼肠道弧菌(Vibrio ichthyoenteri)可引起多种养殖鱼类发病死亡, 给鱼类养殖业带来严重经济损失。为解决养殖过程中鱼肠道弧菌的现场快速检测问题, 本研究研制了鱼肠道弧菌胶体金快速检测试纸。通过制备兔抗鱼肠道弧菌多克隆抗体, 间接ELISA分析发现其与鱼肠道弧菌的外膜蛋白、鞭毛蛋白、胞外产物及全菌破碎蛋白发生阳性免疫反应, 与副溶血弧菌(Vibrio parahaemolyticus)、鳗弧菌(Vibrio anguillarum)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)的4种抗原蛋白发生程度不等的免疫交叉反应。制备胶体金标记鱼肠道弧菌抗体, 确定了鱼肠道弧菌4种抗原蛋白的划线浓度, 以4种抗原蛋白作为4条检测线, 羊抗兔IgG作为质控线, 基于竞争法免疫层析技术研制出鱼肠道弧菌快速检测试纸。对鱼肠道弧菌、副溶血弧菌、鳗弧菌、溶藻弧菌和哈维氏弧菌的检测结果显示, 该试纸可以准确鉴别出鱼肠道弧菌, 并能判别其他病原菌的交叉反应。该试纸对鱼肠道弧菌的最低检测限为5×10⁵ CFU/mL, 检测耗时为10 min。对患病牙鲆组织的检测结果显示, 该试纸与ELISA结果一致, 表明具有较好的检测准确性。本研究为水产养殖现场的鱼肠道弧菌快速、准确检测提供了有力工具。     

沙丁胺醇化学发光免疫检测试剂盒的研制

该研究建立了直接竞争化学发光免疫法检猪尿中沙丁胺醇(Salbutamol,SAL)的方法。并优化了包被抗体浓度、酶标抗原浓度和加样操作模式。优化后的CLIA检测试剂盒,检测范围为0.3~24.3 ng/m L,IC50为0.615ng/m L,理论检测限小于0.1ng/m L。猪尿检测的平均回收率为87±15%,批内相对对标准偏差均小于10%。这些参数表明CLIA检测试剂盒可用于猪尿中沙丁胺醇的检测。     

羊早孕诊断试纸条的研制

用抗孕酮单克隆抗体和羊抗兔多克隆抗体分别包被检测线和质控线,优化了金标抗体、质控线和检测线的包被条件和抗体浓度,应用层析式双抗体夹心法原理研制羊早孕诊断试纸。通过检测尿液中孕酮含量的变化推断是否怀孕,结果显示检出率为47.8%;通过了稳定性和重复性实验,获得了理想的效果,值得在基层推广。     

豚鼠气单胞菌胶体金免疫层析试纸条的研制

采用淋巴细胞杂交瘤技术制备抗豚鼠气单胞菌单克隆抗体分泌细胞株，获取抗豚鼠气单胞菌单克隆抗体，柠檬酸三钠还原氯金酸制备胶体金颗粒，选择直径20 nm的胶体金颗粒，标记抗豚鼠气单胞菌单克隆抗体并制备胶体金垫。将胶体金垫与喷涂有抗豚鼠气单胞菌兔多克隆抗体和羊抗鼠抗体的硝酸纤维素膜及样品吸收垫等组装成免疫层析试纸条，建立豚鼠气单胞菌的快速检测方法。用灭活细菌与血清混合的模拟样本测定试纸条的特异性、灵敏度，结果显示，试纸条对嗜水气单胞菌、温和气单胞菌、鳗弧菌、溶藻弧菌、荧光假单胞菌、副溶血弧菌等13种常见病原菌没有交叉反应，与豚鼠气单胞菌显示特异性反应，检测灵敏度为1×10⁶ CFU/mL，结果显示时间小于5 min。研制的豚鼠气单胞菌胶体金免疫层析检测试纸条具有快速、简便、特异性高、适用于基层临床生产推广应用等优点。     

甲壳类水产品主要过敏原原肌球蛋白胶体金免疫层析快速检测方法的建立及应用

为实现甲壳类水产品主要过敏原的快速检测,实验使用凡纳滨对虾原肌球蛋白(TM)作为检测靶标,构建了胶体金免疫层析检测方法。使用天然TM分别免疫SD大鼠和新西兰大白兔制备多克隆抗体,以40 nm胶体金标记的鼠多抗作为检测抗体,以兔多抗为捕获抗体,基于双抗体夹心原理组装免疫层析试纸条,并应用于市售食物样品的检测。结果显示,组建的免疫层析试纸条可在5 min之内显示结果,视觉检出限为10 ng/mL,对虾、蟹、克氏原螯虾及美洲螯龙虾等甲壳类水产品的特异性高,但与蛤蜊、扇贝存在微弱的交叉反应;在不含甲壳类水产品的市售食品中的加标回收率为81%～126%,准确性良好;试纸条的批内、批间变异系数低于15%,市售实际食物样品的检测结果与食物过敏原标签一致。研究表明,该方法具有高灵敏度与检测特异性,可在多种基质与商业化食品中实现甲壳类过敏原的有效检测,具有较强的实际应用价值。     

水产品中庆大霉素直接竞争酶联免疫吸附测定法的建立

建立了测定水产品中庆大霉素(GM)残留的直接竞争酶联免疫吸附测定法(dcELISA),为进一步开发商业化的ELISA试剂盒提供技术支持。通过碳二亚胺法和戊二醛法分别制备了免疫原GM-KLH和包被原GM-OVA,经动物免疫后获取的多克隆抗体与辣根过氧化物酶偶联制备酶标抗体GM-PAb-HRP。优化确立了dcELISA最佳检测条件:包被原质量浓度0.2μg/m L,GM-PAb-HRP稀释度1/3 200,抗体反应时间40 min,缓冲液为PBS(pH 8.0,10 mmol/L)。该方法的半抑制浓度(IC_(50))为0.99μg/L,定量检测线性范围为0.27~3.64μg/L。水产样品加标回收率为77.7%~104.5%,RSD为6.7%~13.2%,最低检测限为2.0μg/kg。该方法可用于水产品中庆大霉素残留的快速测定,为食品安全风险监测提供一种有效的技术手段。     

抗软骨藻酸单克隆抗体杂交瘤细胞株的构建

为了获得稳定、高效分泌抗软骨藻酸(DA)单克隆抗体的杂交瘤细胞株,采用活泼酯法制备软骨藻酸免疫抗原(DA-KLH)和包被抗原(DA-BSA),以DA-KLH免疫BALB/c小鼠,用细胞融合技术筛选抗软骨藻酸单克隆抗体杂交瘤细胞株,体内诱生腹水法制备大量单抗,捕获EL ISA法测定小鼠免疫球蛋白亚型,间接ELISA法测定腹水效价;用G蛋白亲和层析法来纯化腹水。结果显示,成功构建2株能稳定分泌DA单克隆抗体(McAb)的杂交瘤细胞株2B1和4C2,抗体亚类分别为IgG1和IgG2a,间接EL ISA检测小鼠腹水的抗体效价达1∶64 000,腹水纯化后蛋白浓度为1.27和0.675 mg/mL。研究结果表明,成功制备的抗DA单克隆抗体杂交瘤细胞株稳定、高效,为利用该单抗研制快速检测软骨藻酸的胶体金免疫层析试纸条打下基础。     

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP_AHPND), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb–protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 10⁷ cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VP_AHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.     

Concentration of 17α-Methyltestosterone in HormoneTreated Feed: Effects of Analytical Technique, Fabrication, and Storage Temperature

Feed treated with 17α-methyltestosterone (MT) is used to manipulate the gender of early tilapia fry. In the USA, hormone-treated feed is used by selected producers under an Investigational New Animal Drug (INAD) program. While monitoring the fabrication and on-farm use of this treated feed, concerns were raised about the high performance liquid chromatography (HPLC) analytical procedure for detecting MT in feeds, the incorporation of MT in feeds during fabrication, and effects of storage temperature on MT concentration. A series of experiments demonstrated that the analytical procedure for detecting MT in feeds lacked high precision, and, on average, biased results low. MT was uniformly mixed with feed by spraying an MT-alcohol solution on feed while it was blended in an industrial ribbon mixer. Alcohol volumes ranging from 15 mL/kg to 150 mL/kg were equally effective at dispersing MT in feed. The concentrations of MT in feeds consistently declined over time if the storage temperatures were 25 C or higher. Freezing preserved the MT in feed, and the refrigeration of feed fabricated to contain 60 mg MT/kg maintained acceptable MT concentrations during 6 mo of storage.     

盐酸多西环素临床治疗异育银鲫嗜水气单胞菌感染的给药方案的制定

通过高效液相色谱法(HPLC法)测定盐酸多西环素在异育银鲫(Carassius auratus gibelio)血清中不同时间点的血药浓度,并结合药物的体外药效学参数,确定该药的临床给药方案。结果显示,盐酸多西环素对嗜水气单胞菌(Aeromonas hydrophila,AH10)的最小抑菌浓度(MIC)为0.4μg/m L,最小杀菌浓度(MBC)为3.2μg/m L,防耐药突变浓度(MPC)为4.8μg/m L,耐药选择窗(MSW)为0.4~4.8μg/m L。盐酸多西环素的抗菌后效应(PAE)约为2 h;在异育银鲫中能够达到良好的抑菌杀菌效果的盐酸多西环素口灌剂量是20 mg/kg,休药期至少为25 d;能够控制耐药菌株产生的盐酸多西环素口灌剂量是80 mg/kg,休药期至少为60 d。     

Development of a colloidal gold immunochromatographic assay (GICA) for the rapid detection of Spiroplasma eriocheiris in commercially exploited crustaceans from China

Spiroplasma eriocheiris is an emerging pathogen in freshwater crustaceans. In recent years, Eriocheir sinensis, Procambarus clarkii, Litopenaeus vannamei, Macrobrachium rosenbergii and Macrobrachium nipponense had been infected by this pathogen in China. An immunochromatographic strip test using gold nanoparticles was developed for rapidly detecting this pathogen. The strip test based on the principle of sandwich immunoassay by the specific combination between the pathogen and polyclonal antibody on a nitrocellulose membrane. Positive samples were displayed as red lines at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red line only at the control zone. The limit of detection was proved to be 10⁶ Color Change Unit/ml. The test strip could be visually detected within 15 min and do not have cross‐reaction with other aquatic bacteria. This test strip allows on‐site rapid detection of S. eriocheiris in crustacean without the requirement of specialized equipment and professional personnel. The one‐step test strips developed in our study had high sensitivity, specificity, reproducibility and stability. In conclusion, this method was proved to be convenient, feasible, rapid and effective for detecting S. eriocheiris.     

Maximum limits of organic and inorganic mercury in fish feed

The relatively high levels of mercury found in fish feeds might form a fish health and food safety risk. The present study aims to establish sublethal toxic threshold levels in fish and assess feed‐fillet transfer of dietary mercury. Atlantic salmon (Salmo salar L.) parr were fed for 4 months on fish meal‐based diets supplemented with mercuric chloride (0, 0.1, 1, 10 or 100 mg Hg kg^?1 dry weight (DW)) or methylmercuric chloride (0, 0.1, 0.5, 5 or 10 mg MeHg kg^?1 DW). At the end of the experiment, dietary inorganic mercury mainly accumulated in intestine (80% of body burden) and assimilation was low (6%). In contrast, methylmercury readily accumulated in internal organs and muscle (80% of body burden) and had a relatively high assimilation (23%). Highest accumulation of dietary inorganic mercury was observed in the gut and kidney. Fish fed 10 mg Hg kg^?1 had an early (after 2 months) significant increase in renal metallothionein (MT) level and intestinal cell proliferation, followed by intestinal pathological conditions after 4 months of exposure. At 100 mg Hg kg^?1, intestinal and renal function were reduced as seen from the significantly reduced protein and glycogen digestibility and increased plasma creatinine levels. For dietary methylmercury (MeHg), highest accumulation was found in blood and muscle. Intestinal cell proliferation and liver MT significantly increased at 5 mg MeHg kg^?1 after 2 months of exposure. At the end of the experiment, blood haematology was significantly affected in fish fed 5 mg MeHg kg^?1 and these fish exceeded the current food safety limit for mercury. Tissue MT induction and intestinal cell proliferation appeared to be useful and quantifiable early indicators of toxic mercury exposures. Based on the absence of induction of these early biological markers such as MT and cell proliferation, nonobserved effect levels (NOELs) could be set to 0.5 mg Hg kg^?1 for dietary methylmercury and 1 mg Hg kg^?1 for inorganic mercury. Lowest observed effect levels (LOELs) levels could be set to 5 mg kg^?1 for methylmercury and 10 mg Hg kg^?1 for inorganic mercury.     

Influence of 17-α-Methyltestosterone on Red Tilapia Under Two Thermal Regimes

Fingerling red tilapia ( Oreochromis mossambicus ± O. niloticus ) were orally administered 17-α-methyltestosterone (MT) under a warmwater (27.0 ± 0.5 C) and coolwater (21.5 ± 0.5 C) thermal regime. In the warmwater experiment, fish received either 0, 1, 5, 10, 30, 60, or 100 mg MT/kg feed for 75 days. In the coolwater experiment, fish received 0, 10, or 60 mg MT/kg feed for 75 days followed by a 34 day withdrawal period. After 75 days, fish receiving 60 mg MT/kg feed (best treatment) in the warmwater and coolwater experiments exhibited significantly higher growth rates than controls by 35.3 and 45.8%, respectively. Likewise, feed conversion among groups receiving 60 mg MT/kg feed in the warmwater and coolwater experiments were significantly better than the controls (1.14 versus 1.30 and 1.44 versus 1.77, respectively). During the withdrawal period, no significant differences in growth rates or feed conversion were observed between the control and treatment groups. MT treatment significantly affected the body composition (whole body and empty carcass) of the red tilapia in both experiments, elevating percent moisture and protein values, but depressing percent fat values. Results demonstrated that incorporation of MT into fingerling diets offers potential for extending the period when tilapias actively feed and grow in temperate climates.     

Properties of cod metallothionein,its presence in different tissues and effects of Cd and Zn treatment

One isoform of the low-molecular-weight metal-binding protein metallothionein (MT) has been isolated from the liver of Atlantic cod by size-exclusion and ion-exchange chromatography. Cod MT contained 33% cysteine, no aromatic amino acids or arginine. As is the case for other piscine MTs, the N-terminus of cod MT lacked the asparagine in position 4 which is present in mammalian MTs. In addition, cod MT differed from all other vertebrate MTs described in that the N-terminal methionine was not acetylated. Antibodies were raised in rabbits against hepatic MT from cod by repeated injections of native protein mixed with adjuvant. Anti-cod MT antisera cross reacted with similarly-sized proteins in liver, brain, anterior kidney, posterior kidney, spleen, intestine, gills and ovaries. The putative MT in cod brain migrated differently to that of the other tissues in native gel electrophoresis. Intraperitoneally injected Cd (1 mg/kg) was nearly entirely associated with the MT-peak in hepatic and renal cytosols, whereas a single injection of Zn (10 mg/kg) resulted in increases in all cytosolic Zn pools of the liver and no apparent change in cytosolic Zn, Cu, Ni or Cd in kidney.     

Growth and Bone Development in Channel Catfish Fed 17-α-Methyltestosterone in Production Ponds

Effects of feeding 17-α-methyltestosterone (MT) to channel catfish ( Icralurus punctatus ) grown to harvestable size in earthen ponds were examined. Channel catfish fingerlings (mean weight, 14.4 g), stocked in 0.04 ha ponds (7,410 fish/ha) were fed diets containing MT at concentrations of 0, 2.5, and 10 mg/kg for 123 days. Weight gain by fish fed the control diet (0 MT) was higher ( P < 0.05) than that of fish fed the treated diets. Increasing the dietary concentration of MT reduced weight gain further ( P < 0.05). Both male and female fish fed MT had enlarged and thickened heads, and their skins were dark. Their dorsal and pectoral spines were short and the tips, which are normally very sharp, were soft and blunt. Weight of the rib bones per unit of length decreased ( P < 0.05) as MT was added to the diet. Breaking strength of the ribs (force required to break the bone at its midpoint) measured by an Instron shear press, was less ( P < 0.05) for fish fed MT than for control fish. The ratio of calcium to phosphorus in bones was lower ( P < 0.05) in fish fed MT. These results indicate that feeding MT at these doses (2.5 mg/kg or above) to channel catfish suppresses growth rate and reduces size and strength of bones.     

Human Food Safety and Environmental Assessment of the Use of 17α-Methyltestosterone to Produce Male Tilapia in the United States

Sex reversal of early life stage tilapia (approximately 7–12 d post-hatch, total length averaging 9–11 mm, and total weight averaging 10–15 mg) is used commonly to produce populations of fish comprised of > 5% phenotypic females. The synthetic androgen, 17α-methyl testosterone (MT), is used to effect sex reversal in tilapia. This paper evaluates environmental impact and human food safety aspects of MT use for tilapia sex reversal based on a review of the scientific literature and on dilution models of farm discharge.
Effect of MT treatment on human food safety was evaluated by regression analysis of radioactivity depletion data and by dilution through growth analysis. Results show that the proposed use of MT for sex reversal of early life stage tilapia presents no negative effects on human food safety. Regression analysis of available depletion data from tilapia shows that whole-body concentrations of MT and metabolites in tilapia attain levels of <100 pg/g after 8 to 40 d of withdrawal, and that achievement of <10 pg/g of MT and metabolites in carcass tissue occurs after 6 to 50 d of withdrawal. These concentrations are attained well before completion of the FDA-required 120-d withdrawal period following MT treatment.
Standardized calculations were made to estimate MT concentration in effluents based on stocking rate and water exchange. Guidelines are provided for management of farms to ensure that effluent MT concentration remains < 1 μg/L. Given the expected concentration of MT in any farm effluents, the rapid dilution of effluent MT to very low levels in receiving waters, the sensitivity of MT to photo-oxidation, the expected rapid bacterial degradation of MT, and the limited total use of MT in the United States, we concluded that the use of MT-treated feed according to the proposed protocol will not cause significant adverse environmental effects.