血清结合甘胆酸放射免疫分析药盒研制(阻断法)

人血清中结合甘胆酸含量测定,采用阻断法放射免疫分析药盒,达到直接测定血样中结合甘胆酸的含量。本药盒以~(125)I-甘胆酸组胺为标记抗原,放化纯>90%。用去激素正常人血清配制系列标准,标准浓度分别为0.25.100.250.1000.4000μg/dl。兔抗血清,16~#兔抗血清亲和常数K=2.6×10~8l/mol。以0.5%ANS(8-苯胺-1-萘磺酸)磷酸盐缓冲溶液为阻断剂。PEG(聚乙二醇)为分离沉淀剂。本药盒分析灵敏度10μg/dl。批内批间精密度分别为4.7%和12.5%。平均回收率109%。     

重组鲈鱼生长激素的分离纯化及抗体制备

用IPTG诱导鲈鱼生长激素基因工程菌E.coli.RV308,使重组鲈鱼生长激素得以表达并分泌到周质空间,利用渗透休克法从菌液中提取间质蛋白,经DEAE-SepharoseCL-6B、矣丙烯酰胺凝胶电泳及SephadexG-75纯化后得到单一电泳纯的重组鲈鱼生长激素,并将此激素作为抗原制备抗体,可应用于鱼类血液中生长激素含量的测定。     

Development of competitive enzyme-linked immunosorbent assays for boscalid determination in fruit juices

Boscalid is a modern, broad-spectrum carboxamide pesticide highly efficient against most fungal diseases affecting valuable crops. In this study, a boscalid-mimicking derivative with a six-carbon spacer arm replacing the chlorine atom at the pyridine ring of the target molecule was synthesized and coupled to carrier proteins. Following rabbit immunization, antibodies against this agrochemical were obtained for the first time, and they were characterised in terms of affinity and specificity, tolerance to solvents, and robustness to changes in buffer pH and ionic strength, using two assay formats. Both of the optimised immunoassays showed limits of detection below 0.1 μg/L. Moreover, matrix effects of grape, peach, apple, and tomato juices were evaluated. Finally, a simple and easy procedure was set up for boscalid determination with spiked samples, affording limits of quantification of 10 μg/L, a value well below the sensitivity levels required for monitoring campaigns of pesticide residue analysis in food.     

副溶血性弧菌诊断血清的比较

目的比较我国自主生产和进口的副溶血性弧菌诊断血清在检出率、反应强度和特异性等方面的差异。方法使用进口和我国自主生产的诊断血清同时对35株副溶血性弧菌标准菌株和60株临床菌株进行玻片凝集,以检测其血清型。结果对于参考菌株,两者的检出率均为100%,对于临床株,两者的一致率为95%;且两者的特异性均良好。结论我国自主生产的副溶血性弧菌诊断血清性能良好,可以用于副溶血性弧菌分型鉴定。     

克喘素免疫昆明鼠产生多抗血清效果的初步研究

用克喘素(CL)与牛血清白蛋白(BSA)偶合制备的分子结合比为13.2:1的克喘素免疫原CL13.2-BSA免疫昆明鼠,制备多抗血清,采用间接ELISA和间接竞争ELISA法检测,进行克喘素的昆明鼠多抗血清效果的初步研究,结果表明:昆明鼠产生多抗血清的平均效价为40000,对克喘素的线性检测范围为10ng/mL~1礸/mL。     

甘胆酸放射免疫分析的几个问题

本文讨论了甘胆酸组胺(CGH)的碘化标记,单~(125)I-CGH免疫活性(B_0/T=70%)>双~(125)I-CGH免疫活性(B_0/T=45%)。对甘胆酸抗血清质量(亲和常数K,交叉反应率和滴度)作了研究。从logit-log线性计算结果指出,抗血清质量影响着放免分析的测定。保护蛋白影响着抗血清结合率。CG标准采用特殊的去激素血清配制。     

人乳头瘤病毒58型截短型L1蛋白与结核分枝杆菌早期分泌型抗原靶蛋白-6的融合表达及其抗血清的制备

目的原核表达人乳头瘤病毒(Human papilloma virus,HPV)58型截短型L1(Truncated L1,tL1)蛋白与结核分枝杆菌早期分泌型抗原靶蛋白-6(Early secreted antigenic target-6,ESAT-6)融合蛋白,并制备其抗血清。方法利用PCR技术分别从HPV基因组DNA和ESAT-6-18T质粒DNA中扩增tL1和ESAT-6基因,构建重组原核表达质粒ESAT-6-tL1-pET-21a,分别转化E.coli BL21(DE3)和Rosetta,IPTG诱导表达。表达的融合蛋白ESAT-6-tL1经镍离子亲和层析柱纯化后免疫昆明小鼠,ELISA检测血清抗体效价。结果 PCR扩增出750 bp的tL1基因片段和320 bp的ESAT-6基因片段,测序结果与GenBank登录的序列一致;重组表达质粒ESAT-6-tL1-pET-21a经双酶切鉴定,构建正确;表达的ESAT-6-tL1融合蛋白相对分子质量约40 000,在E.coli Rosetta中的表达形式为包涵体,纯化后纯度为85%,免疫小鼠血清抗体效价为1:4 000。结论成功表达了ESAT-6-tL1融合蛋白,并制备了其抗血清,为进一步研制检测HPV58的抗体奠定了基础。     

小鼠载脂蛋白A-Ⅰ的原核表达及抗血清的制备

目的原核表达小鼠载脂蛋白A-Ⅰ(ApoA-Ⅰ),并制备抗血清。方法应用RT-PCR方法从小鼠肝脏中扩增ApoA-Ⅰ基因,亚克隆至原核表达载体pET-28a(+),转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的融合蛋白经Ni2+-NTA柱纯化后,免疫家兔,制备抗血清。结果扩增的ApoA-Ⅰ基因经测序,表明与GenBank中报道的序列(NM_009692)完全相同。重组表达质粒经酶切鉴定,证明构建正确。表达的ApoA-Ⅰ融合蛋白相对分子质量约为32000,表达量占菌体总蛋白的17%。纯化的融合蛋白纯度达90.7%,可与抗His·Tag单抗发生特异性反应。制备的抗血清可识别ApoA-Ⅰ融合蛋白,效价可达1∶105。结论已在大肠杆菌中表达了小鼠ApoA-Ⅰ,并制备了较高效价的抗血清。     

中蜂囊状幼虫病病毒结构蛋白的表达及其抗血清的制备

目的原核表达中蜂囊状幼虫病病毒(Chinese sarbood virus,CSBV)结构蛋白,并制备其抗血清。方法采用RT-PCR法扩增CSBV结构蛋白基因片段,克隆至pGEX-4T-1载体中,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经Gluthathione-Sepharose 4B亲和层析纯化后,免疫小鼠,制备抗血清,并进行Western blot鉴定。结果重组表达质粒经菌落PCR鉴定证明构建正确;表达的重组蛋白相对分子质量约52 000,以可溶性形式表达;纯化后纯度可达95%以上;制备的抗血清可识别天然CSBV结构蛋白。结论成功在大肠杆菌中表达了CSBV结构蛋白,并制备了其抗血清,为进一步研究囊状幼虫病病毒与宿主的相互作用及开发血清学诊断方法奠定了基础。     

Identification of fish species in dried fish products by immunostaining using anti-myosin light chain antiserum

In order to immunologically detect fish species used in boiled and dried fish products (dried fish sticks), the availability of antiserum against myosin light chains was examined. After dissolving the samples in the presence of 8 M urea and 1% sodium dodecyl sulfate (SDS), the solubilized matters were applied to SDS–polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Subsequently, protein bands concerned were stained using anti-myosin light chain (alkali light chain 1) rabbit antiserum and horseradish peroxidase-conjugated anti-rabbit immunoglobulin. Most of the fish species could be identified by fingerprinting of immunostained patterns even after drastic processing, even though the protein staining patterns were not clear enough for species identification.