动物源性食品中泰乐菌素残留ELISA试剂盒的研制

采用杂交瘤技术,筛选并克隆出能够稳定分泌抗泰乐菌素（TYL）单抗的杂交瘤细胞株,免疫BALB／c小白鼠制备抗泰乐菌素单克隆抗体,建立泰乐菌素竞争ELISA检测试剂盒。结果表明,logit／log拟合标准曲线为y=-1．84x＋2．02,相关系数r=0．9944,线性检测范围为1．5～121．5ug／L,半数抑制浓度（IC50）为10.3ug／L,灵敏度为1．5ug／L,检测限为1．5～3．0ug／L;肌肉、肝脏、蜂蜜的添加回收率分别为64％～99％、61％～102％、60．5％～95％;平均批内和批间变异系数均小于10％;泰乐菌素与替米卡星（TIL）的交叉反应为40％,与其他类抗生素无交叉反应;此泰乐菌素试剂盒在4℃可保存6个月以上。本文建立的竞争ELISA检测方法可用于动物源性食品中泰乐菌素残留的定量检测。     

鲤疱疹病毒Ⅱ型ORF4基因的克隆、表达与免疫学检测方法

根据鲤疱疹病毒II型(Cyprinid herpesvirus Ⅱ,Cy HV-2)ORF 4基因序列(Gen Bank:JQ815364.1)设计特异性引物,PCR扩增得到ORF 4基因编码框全长序列1 041 bp,将其克隆至原核表达载体p ET-32a(+)中,构建了重组原核表达载体p ET-32a-ORF 4。将p ET-32a-ORF 4重组载体转化大肠杆菌BL21(DE3),经IPTG诱导得到融合表达的重组蛋白,融合表达的重组蛋白主要以包涵体的形式存在,其分子质量约为57 ku,与预期大小一致。将纯化的重组蛋白免疫日本大耳兔,制备了多克隆抗体,ELISA检测抗体效价大于1∶50 000,Western blot检测显示该抗体可以特异性识别重组蛋白。间接免疫荧光检测结果表明:该多克隆抗体可与由Cy HV-2感染引起细胞病变的异育银鲫脑组织细胞(GICB)发生特异性的结合。     

Cutaneous antibodies from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis (Ich) may induce apoptosis of Ich theronts

This study explored the existence of apoptosis (programmed cell death) in Ichthyophthirius multifiliis Fouquet (Ich) theronts and determined the effect of cutaneous antibodies in skin culture fluid from fish immune to Ich on theront apoptosis. Apoptosis was detected in theronts and was clearly distinguished by fluorescent microscopy after staining with acridine orange and propidium iodide. The apoptotic theronts showed characteristic chromatin condensation and nuclear fragments containing chromatin pieces. The externalization of phosphatidylserine on the plasma membrane of apoptotic theronts was detected with fluorescein isothiocyanate-conjugated annexin using flow cytometry. Theront apoptosis was induced using the skin culture fluid from fish immune to Ich, which contained cutaneous antibodies against Ich. The highest apoptosis appeared in theronts exposed to immune skin culture fluid at a 1:10 dilution, compared with those at 1:20 and 1:40 dilutions. A direct correlation was noted between the percentage of apoptotic theronts and exposure duration to immune skin culture fluid. The study indicated that antibody reaction with theronts (immobilization) played an important role in theront apoptosis, but it could not be excluded that other components released from the excised skin had effects on theronts.     

Influence of size, age and maternal condition on the oocyte dry weight of Argentine hake (Merluccius hubbsi)

Effects of maternal age–size and condition on oocyte dry weight were analysed for Merluccius hubbsi. Samples of mature females were collected in the Patagonian spawning area during 10 research surveys carried out between 2000 and 2005. Dry weight of 50 hydrated oocytes ranged from 0.80 to 1.90 mg. Relationships of this variable with total length and age were best described by a logarithmic model during the spawning peak. At the beginning and at the end of the reproductive season no significant relationship between these variables was observed, but a decrease in egg dry weight was detected during the spawning period. In general, young spawners (≤5-year-old) showed more variability in egg dry weight, which was associated with a reproductive season shorter than that of old females. Relative condition factor did not shows a significant relationship with oocyte dry weight, and mean values of this index were similar for different months of the spawning seasons analysed.     

抗Asia1型口蹄疫病毒单克隆抗体的制备及抗原捕获ELISA检测口蹄疫病毒的研究

本研究用纯化的Asial型口蹄疫病毒(Foot and mouth disease virus,FMDV)免疫BALB/c小鼠,按常规单克隆抗体技术方法,经筛选获得6株能稳定分泌抗Asial型FMDV单抗的杂交瘤细胞株。以牛抗口蹄疫病毒IgG为捕获抗体,选择一株单抗(184)用辣根过氧化物酶标记(HRP-184)作为检测抗体,建立了检测Asial型FMDV的抗原捕获ELISA方法。该方法可检出0.5859μg纯化Asial型FMDV抗原和2.5×10~3TCID_(50)病毒,对O型FMDV、牛结核病、牛肺疫、牛流热、赤羽病、牛传染性鼻炎等病毒进行检测,均为阴性,无交叉反应发生。本研究建立的FMDV抗原捕获ELISA方法,具有敏感性高、特异性强和重复性好的特点,可用于Asial型FMDV的特异性检测。     

抗氯霉素单克隆抗体的制备及其化学发光酶免疫法的建立

以人工合成的氯霉素-牛血清白蛋白(CAP-HS-BSA)免疫Balb/c小鼠,应用杂交瘤技术获得了一株分泌抗氯霉素单克隆抗体(CAP-McAb)的杂交瘤细胞5D7,并对其进行效价、亲和力和特异性测定。结果显示,其分泌的抗体亚类为IgG1,间接ELISA检测细胞培养上清效价为1∶512,诱生的腹水经纯化后效价为1∶2×105,亲和常数为3.6×108L/mol;应用上述单抗建立的检测氯霉素的化学发光酶免疫分析法(ci-CLIA),其检测范围为0.001μg/L~10μg/L,检出限达0.0025μg/L,IC50为39.6μg/L,且与L-苯丙氨酸、L-酪氨酸、青霉素、四环素、庆大霉素等的交叉反应率均小于0.01%,可满足我国对水产品中氯霉素残留检测的要求。     

一种免疫增强剂对新-流二联灭活苗抗体消长规律的影响

免疫增强剂是一种能增加疫苗免疫效果的生物活性物质。试验A组为添加了免疫增强剂的新城疫、禽流感(H9亚型)二联灭活疫苗(La Sota株+HP株),试验B组为未添加免疫增强剂的新城疫、禽流感(H9亚型)二联灭活疫苗(La Sota株+HP株),分别比较A组与B组的抗体水平。试验结果表明:7日龄SPF鸡A组抗体与B组抗体无差异;在28日龄SPF鸡上:免后10、15、21、28、35 d,ND部分A组抗体比B组分别高0.2log2、0.6log2、1.5log2、0.9log2、0.5log2,AI部分抗体高出0.1log2、0.3log2、1.2log2、1.0log2、0.6log2;在7日龄商品蛋鸡上A组抗体比B组抗体在免后15、21、28、35 d,ND部分抗体高出1.6log2、2.0log2、1.9log2、3.7log2,AI部分分别高1.3log2、2.7log2、1.8log2、2.0log2。     

旋毛虫肌幼虫ES Ag单克隆抗体的制备与特性鉴定

目的获得能够用于制备旋毛虫病快速检测试纸条的EsAg的单克隆抗体。方法应用旋毛虫肌幼虫排泄分泌抗原（ESAg）免疫诱导Balb／c小鼠,使小鼠产生较强的免疫应答,将免疫小鼠的脾细胞与NS0瘤细胞融合,利用Es45、ES49抗原通过间接ELISA对大量杂交瘤细胞培养上清的筛选,筛选出分泌高亲和力单克隆抗体的4株杂交瘤细胞。结果ES45和ES49（分子质量分别为45ku,49ku）分别被Ts-2D4、Ts-4C5和Ts-4H6、Ts-2G8单克隆抗体识别,与猪肺丝虫（Metastrongylus pudendotectus．MP）、囊虫（Cysticercus cellulosae,CC）、细颈囊尾蚴（Cysticercus tenuicollis,CT）、蛔虫（Ascaris suum,AS）、弓形虫（Toxoplasma gondii,TG）、住肉孢子虫（Sarcocystis miescheriana,SM）抗原反应测试表明,4株单抗与参试抗原均无交叉反应,所有单抗上清ELISA平均效价为1：1120,腹水ELISA平均效价为1．1×10^6,亲和力常数的平均值为6．12×10^9 L／mol。以金标免疫吸附试纸条原理为基础,利用制备的单抗成功研制了旋毛虫病快速检测试纸务,操作快速、无需设备及试剂,可以作为旋毛虫病的实时监测工具。结论ESAg单抗用于制备旋毛虫病快速诊断试纸条是理想的试剂。     

抗犬瘟热病毒重组核衣壳蛋白单克隆抗体的制备及鉴定

以纯化的重组犬瘟热病毒(CDV)N蛋白免疫BALB/c小鼠,应用常规杂交瘤技术获得两株能稳定分泌特异性单克隆抗体的杂交瘤细胞系,分别命名为A_4C_6C_(12)和A_5B_8H_7。间接ELISA检测腹水效价分别为1:10~6、1:10~5;亚类鉴定结果分别为IgG2a、IgG2b,轻链均为κ型;Western blot和ELISA分析结果显示2株单克隆抗体均能与重组N蛋白和CDV发生反应,而与犬细小病毒及犬腺病毒等无交叉反应;ELISA叠加试验的增值结果表明两株单克隆抗体识别的抗原位点不同。特异性抗CDV-rN的单克隆抗体的获得,为进一步用于临床诊断研究奠定了基础。