梭鱼卵黄蛋白原的化学发光免疫检测法

运用建立化学发光免疫(chemiluminescence immunoassay,CLIA)检测法,对梭鱼血清中主要卵黄蛋白原(Vg)类型(B类型;VgB)进行定量测定。梭鱼VgBCLIA法按两步法操作进行,使用纯化的梭鱼VgB制备特异型抗血清(a-VgB)。优化抗体浓度和培育时间等检验条件后,检验范围设定为3.91～500ng/mL。卵黄发生期的梭鱼雌鱼血清和雌激素处理后的梭鱼稚鱼血清稀释曲线与纯化的梭鱼Vg曲线平行,而梭鱼雄鱼稀释血清中几乎不发生相应的免疫学反应。对天津原种场养殖的10尾梭鱼血清VgB水平的定量检测结果表明,在雌鱼(9尾)血清中检测到VgB但个体间差异较大,变化范围为3.0～2700.1μg/mL,并表现出随着卵巢发育而增长的变化趋势。相对而言,成熟梭鱼雄鱼血清VgB水平则极低(2.7μg/mL),表明在其生长环境中不存在雌激素活性。另外,所有个体的组织学观察也未呈现出明显的性腺异常情况。研究为梭鱼主要雌激素诱导生物标志物(VgB)的量化提供了一种新的手段,较以往的检验方法具有更高的灵敏度,有助于建立多元化的水生环境中雌激素活性的检测体系。     

日本鳗鲡胶原蛋白和小清蛋白的过敏原性

以日本鳗鲡皮与肌肉组织为研究对象,采用碱溶、酸溶、盐析、冻干等方法纯化得到胶原蛋白,采用加热、饱和硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析等方法纯化得到两种亚型的小清蛋白(PV-Ⅰ和PV-Ⅱ),纯化的目标蛋白经动物特异性抗体的免疫印迹实验确证。酶联免疫吸附测定和免疫印迹分析结果显示,纯化的胶原蛋白和小清蛋白分别与鱼类过敏患者阳性血清发生特异性反应,且二者之间无免疫交叉反应。体外模拟胃液消化实验和SDS-PAGE分析结果显示,胶原蛋白和小清蛋白均具有较高的消化稳定性。结果提示,日本鳗胶原蛋白和小清蛋白具有较高的消化稳定性和免疫原性,二者可引发不同患者的IgE介导特异性超敏反应。     

酶联免疫法测定辣椒中辣椒素含量

旨在建立一种简便快速低成本的辣椒素测定方法。辣椒样品中辣椒素经69.15%乙醇,振摇10min,离心5min,取上清液100μL,置于2.0mL样品瓶中,加0.9mL去离子水,旋涡混匀后用于酶标仪分析。结果表明：标准曲线满足线性方程Y=-42.9X+30.3,相关系数R2=0.9999;在100mg/100g添加水平下,平均回收率测为92.6%,重复性相对标准偏差小于3.5%;方法检出限为0.25mg/100g。300余份新鲜辣椒样品中辣椒素含量测定结果在1.50~323.8mg/100g之间。采用酶联免疫法测定辣椒中的辣椒素含量,有操作简单、测定速度快等优点,尤其适合批量样品的测定。     

化学发光直接竞争免疫分析法检测猪尿中莱克多巴胺残留

本试验研究建立了猪尿中莱克多巴胺（Ractopamine, RAC）残留的直接竞争化学发光免疫检测方法。合成了莱克多巴胺和辣根过氧化物酶的偶联物（RAC-HRP）,方法的检测限为0.09 ng/mL,空白猪尿中添加浓度为0.5、10和100 ng/mL RAC时,检测范围为0.3～157.0 ng/mL;回收率为76.3%～87.6%,批内变异系数为10.0%～12.2%,批间变异系数为14.2%～15.2%。     

Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy

Rabbit sera (n = 1600) from 40 commercial farms were submitted to a serological screening for Encephalitozoon cuniculi by an enzyme-linked immunosorbent assay (ELISA) and a carbon immunoassay (CIA test). Antibodies anti-Encephalitozoon cuniculi were found in 505/1600 (31.6%) sera analysed, and all the farms (100%) resulted positive. Rabbits older than 4 months showed a significantly higher seropositivity for E. cuniculi (chi-squared test: p < 0.0001) than rabbits under 4 months, E. cuniculi sero-prevalence showed an increasing trend in rabbits within the farm along with the increase in the “number of rabbits on the farm”; however, this trend was not significant (Spearman’s correlation: p = 0.073).The findings of the present study confirm that rabbit is the main reservoir of E. cuniculi; they are of epidemiological relevance and immediate public health importance because of the recognized infectivity in humans by the microsporidium.     

Comparison of the VIDAS and IMMULITE‐2000 methods for cortisol measurement in canine serum

Background: Serum cortisol concentration is often measured in dogs for the diagnosis and monitoring of adrenal disease. An enzyme‐linked fluorescent assay (VIDAS method) on the MiniVidas analyzer has been validated for the measurement of cortisol concentration in human serum and could have applications for canine samples. Objective: The aim of this study was to compare canine cortisol results obtained using the VIDAS method with those obtained using the IMMULITE‐2000 immunoassay, which has previously been validated for canine serum. Methods: The concentration of cortisol in 40 canine serum samples was determined concurrently with the VIDAS and IMMULITE methods, the latter as the reference method. Pearson's correlation coefficient, linear, and Deming regression analyses and Bland–Altman analysis were used to compare the 2 methods. Acceptability of the new method was judged using a medical decision chart (MEDx chart). Results: Cortisol concentrations obtained with the IMMULITE method ranged from 23.1 to 1380 nmol/L. Correlation (r=.977) and simple linear regression (slope=1.0722, confidence interval [CI] 0.996–1.148; intercept=?4.799, CI ?42.838 to 33.240) revealed no proportional or constant error. Based on Deming regression and a Bland–Altman plot the 2 methods gave comparable results. The MEDx chart indicated that performance of the new method was good at decision limits of 40, 132, and 480 nmol/L. Conclusion: Results of the VIDAS method were comparable to those of the IMMULITE‐2000 reference method such that the VIDAS may be used as an alternative assay to evaluate serum cortisol concentration in dogs for the diagnosis of adrenal disease.     

四环素残留的免疫学检测方法研究进展

四环素类抗生素具有广谱抗菌能力,因而在畜牧养殖业中被大量使用,但如果不规范用药,就会在动物性食品中残留。传统检测方法检测程序繁琐,检测耗时耗力,设备昂贵,难以实现在线、实时、大批量的检验需求,免疫学检测方法则可弥补这些缺点。作者主要对四环素的残留、危害及免疫检测方法进行了综述。     

Development of a monoclonal antibody‐based flow‐through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

A flow‐through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL^?1 crude WSSV protein, respectively. The FTA could be completed in 8–10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1‐step polymerase chain reaction (PCR) and in between that of the 1‐ and 2‐step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post‐larvae, WSSV was first detected 14, 16 and 18 h post‐infection (hpi) by FTA, immunodot and one‐step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one‐step PCR, respectively. The FTA was more sensitive (25/27) than one‐step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4–8 °C. The FTA is available as a rapid test kit called ‘RapiDot’ for the early detection of WSSV under field conditions.     

禽流感病毒检测技术的研究新进展

禽流感已被列为世界卫生组织(WHO)的流感大流行警告5级,中国将其列为一类传染病。该病的暴发不仅对养禽业造成毁灭性的危害,而且严重威胁到人类生命安全。为此快速准确的诊断对禽流感的防控具有重大的意义,文章就禽流感诊断方法的研究进展作一综述。     

用化学发光免疫法检测有机氯农药DDTs的残留量

建立了检测有机氯农药DDTs残留的化学发光免疫分析技术（chemiluminescence immunoassay,CLIA）,以鲁米诺和过氧化氢作为化学发光底物,测定样品中有机氯农药DDTs残留。结果表明：发光底物液非催化下,5～7 min后发光值较稳定;在发光底物液酶催化下,反应10 min接近峰值;DDTs标准曲线线性范围为0.05～25 ng/mL,标准样品DDTs浓度与发光值呈现很好的线性相关,线性方程为y=-63.806x＋13426,R2=0.9945,检测最低限为0.05 ng/mL;回收率为91.4%～107.8%。