Purification of chinook salmon (Oncorhynchus tshawytscha) GH for receptor study

A method for the purification of chinook Salmon (Oncorhynchus tshawytscha) GH, which retains its biological activity, is described. The biological activity was investigated with an established radioreceptor assay using liver membranes from pregnant rabbits and bovine GH as standard and labelled hormone. The enrichment of the preparation was checked with electrophoresis (SDS-PAGE). Extraction and further steps were carried out using low molarity alkaline buffer (pH 8–10, M = 100 mM). Three chromatography steps were performed (Concanavalin-A sepharose, Bio-gel P60, DEAE). Ion exchange chromatography was performed under isocratic conditions (using a 50 cm column). Two isoforms (sGH1 and sGH2) were isolated. The purification yield is 0.7% compared to lyophilized pituitaries. The molecule is homogeneous in SDS-PAGE. Contamination by prolactin, gonadotrophin and corticotrophin is negligible (< 0.5%). It could be demonstrated that the biological activity of the preparation is maintained since this preparation stimulates the growth of juvenile trout (Salmo gairdneri) and binds specifically (35%) to trout liver membranes.     

Mechanochemical properties of ordinary and dark muscle myosins from seawater fish

Myosin was isolated from two types of muscle, ordinary and dark muscles, of three species of fish living in sea water. The compositions of light chains were visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the mechanochemical activity was examined by in vitro motility and ATPase assays. Ordinary muscle myosin of either species had three species of light chain, whereas dark muscle myosin had another two species of light chain judged by SDS-PAGE. Sliding velocity of ordinary muscle myosin was in the range of 4.92–6.89 μm/S, whereas that of dark muscle myosin was in the range of 3.07–4.25 μm/s. Therefore, ordinary muscle myosin showed 1.26–1.95 times higher sliding velocity than dark muscle myosin in either species. The ratios of V_max of actin-activated Mg²⁺-ATPase activity of ordinary to dark muscle myosins were correlated quite well to the ratios of sliding velocity. Activity of ordinary muscle myosin was comparable to that of mammalian fast muscle myosin, but that of dark muscle myosin was twice of that of mammalian slow muscle myosin. These results may reflect the essential role of fish dark muscle myosin always used in slow cruising.     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.     

Simple and Rapid Quality Control of Sulfated Glycans by a Fluorescence Sensor Assay—Exemplarily Developed for the Sulfated Polysaccharides from Red Algae Delesseria sanguinea

Sulfated polysaccharides (SP) from algae are of great interest due to their manifold biological activities. Obstacles to commercial (especially medical) application include considerable variability and complex chemical composition making the analysis and the quality control challenging. The aim of this study was to evaluate a simple microplate assay for screening the quality of SP. It is based on the fluorescence intensity (FI) increase of the sensor molecule Polymer-H by SP and was originally developed for direct quantification of SP. Exemplarily, 65 SP batches isolated from the red alga Delesseria sanguinea (D.s.-SP) and several other algae polysaccharides were investigated. Their FI increase in the Polymer-H assay was compared with other analytical parameters. By testing just one concentration of a D.s.-SP sample, quality deviations from the reference D.s.-SP and thus both batch-to-batch variability and stability can be detected. Further, structurally distinct SP showed to differ in their concentration-dependent FI profiles. By using corresponding reference compounds, the Polymer-H assay is therefore applicable as identification assay with high negative predictability. In conclusion, the Polymer-H assay showed to represent not only a simple method for quantification, but also for characterization identification and differentiation of SP of marine origin.     

基于量子点的赭曲霉毒素AsFLISA检测技术研究

建立了基于量子点标记技术的赭曲霉毒素A(Ochratoxin A,OTA)双抗夹心荧光免疫检测(sandwich fluorescence-linked immunosorbent assay,sFLISA)体系,包含多克隆包被抗体、生物素标记的多克隆检测抗体、量子点标记的链霉亲和素等,获得了sFLISA的最佳操作参数:包被抗体浓度2.5μg/mL,检测抗体稀释500倍,量子点标记链霉亲和素稀释100倍。该方法在OTA浓度3.125~125μg/L之间时,相对荧光强度和OTA浓度呈线性关系,回归方程为y=0.0206x+0.2018,R2=0.9924;加标回收率在90.1%~110.0%之间,变异系数均小于10%,能较好地进行赭曲霉毒素A的定量检测。     

Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida

Nanoparticles (NPs) of TiO₂ and ZnO are receiving increasing attention due to their widespread applications. To evaluate their toxicities to the earthworm Eisenia fetida (Savigny, 1826) in soil, artificial soil systems containing distilled water, 0.1, 0.5, 1.0 or 5.0 g kg⁻¹ of NPs were prepared and earthworms were exposed for 7 days. Contents of Zn and Ti in earthworm, activities of antioxidant enzymes, DNA damage to earthworm, activity of cellulase and damage to mitochondria of gut cells were investigated after acute toxicity test. The results from response of the antioxidant system combined with DNA damage endpoint (comet assay) indicated that TiO₂ and ZnO NPs could induce significant damage to earthworms when doses were greater than 1.0 g kg⁻¹. We found that Ti and Zn, especially Zn, were bioaccumulated, and that mitochondria were damaged at the highest dose in soil (5.0 g kg⁻¹). The activity of cellulase was significantly inhibited when organisms were exposed to 5.0 g kg⁻¹ of ZnO NPs. Our study demonstrates that both TiO₂ and ZnO NPs exert harmful effects to E. fetida when their levels are higher than 1.0 g kg⁻¹ in soil and that toxicity of ZnO NPs was higher than TiO₂.     

酶联免疫吸附分析技术在农药残留分析中的应用

简述酶联免疫吸附分析技术的原理、方法类型以及关键的技术要点,并对该技术在农药残留分析检测中的应用前景进行了展望。     

雏鸡感染巨型艾美耳球虫后细胞免疫的研究

通过用地塞米松处理鸡和用特异性抗原刺激因子的微量全淋巴细胞转化试验（ＬＰＡ），研究雏鸡抗Ｅｉｍｅｒｉａｍａｘｉｍａ再感染中细胞免疫现象。结果表明，地塞米松处理可损伤鸡抗Ｅ．ｍａｘｉｍａ再感染的免疫力，这提示了ＣＤ８＋Ｔ细胞，γ－ＩＦＮ及ＩＬ－２等在抗．Ｅｍａｘｉｍａ再感染中的作用，利用Ｅ．ｍａｘｉｍａ卵囊抗原作刺激因子的微量全血ＬＰＡ结果表明，其刺激指数值与鸡抗Ｅ．ｍａｉｘｉｍａ再感染抵抗力有关。     

新生牛血清促细胞生长活性检测方法的建立与应用

牛血清是医药生物技术产品中重要的原辅材料之一,做好牛血清的质量控制是提高生物制品质量的重要环节。本研究通过应用PK15、BHK21和ST三种兽用生物制品研制中常用的细胞作为载体,选用国内同等价位5个不同厂家生产的新生牛血清作为比较对象,采用细胞倍增时间测定法、细胞克隆形成率测定法、细胞三次连续传代培养法、MTT比色法和微量终点稀释法五种血清质量鉴定的方法进行比较,检测新生牛血清的促细胞生长活性,最终确定了MTT比色法作为便捷高效快速筛选新生牛血清的检测方法。     

犬瘟热病毒抗体检测方法的比较研究

用犬瘟热（ＣＤ）弱毒和自制的高免犬血清，建立了检测犬瘟热病毒（ＣＤＶ）抗体的中和（ＳＮ）试验、间接荧光抗体技术（ＩＦＡＴ）和间接免疫酶染色法。这３种方法对３８份ＣＤ弱毒疫苗免疫犬血清抗体效价的测定结果表明，当被检血清稀释度为１∶１００时，ＳＮ试验、ＩＦＡＴ和间接免疫酶染色法测定时的阳性血清数分别为２９，７和３１份，ＩＦＡＴ和间接免疫酶染色法测定结果相对于ＳＮ试验结果的符合率分别为４２．１％和９４．７％。同时发现，当ＳＮ试验测定的血清效价在１∶１００以上时，ＩＦＡＴ测定结果总是在１∶２５以上，间接免疫酶染色法测定结果总是在１∶１００以上。３种方法以各自初步确定的ＣＤＶ血清抗体效价完全保护值指标计算所测血清的完全保护率，ＳＮ试验为７６．３％，ＩＦＡＴ为７８．９％，间接免疫酶染色法为８１．６％。据此认为，ＩＦＡＴ和间接免疫酶染色法均可部分代替ＳＮ试验用于ＣＤ疫苗免疫效果的评价、免疫犬抗体水平的监测以及ＣＤ的流行病学调查