抗ICAM-1单克隆抗体的制备及其活性

目的制备具有生物活性的抗人细胞间黏附分子-1(ICAM-1)单克隆抗体。方法以人ICAM-1为抗原免疫BALB/c小鼠,采用杂交瘤技术并经HAT选择培养和克隆化,筛选出稳定分泌抗人ICAM-1 McAb的杂交瘤细胞株,并对McAb进行纯化。用ELISA间接法测定效价并鉴定其亚类,Western blot鉴定其抗原特异性,细胞黏附试验检测其中和活性。结果筛选出1株可稳定分泌抗人ICAM-1抗体的杂交瘤细胞株3F2,杂交瘤染色体众数为98~104。纯化后的单抗蛋白浓度为1.253 mg/ml,纯度达83.6%,效价可达2.56×105。其分泌的抗体亚型为IgG1,腹水效价达5.12×105,可与ICAM-1特异性结合,可抑制ICAM-1与淋巴瘤细胞间的黏附,具有明显的中和ICAM-1的活性。结论已成功制备出抗ICAM-1的McAb,为其进一步的研究和应用奠定了基础。     

幽门螺杆菌尿素酶B亚单位特异性抗体检测方法的建立与评价

目的建立并评价幽门螺杆菌(Hp)尿素酶B亚单位(UreB)特异性抗体的检测方法。方法应用纯化的重组HpUreB作为包被抗原,建立检测HpUreB特异性抗体的ELISA间接法。以Hp抗体Western blot检测作为“金标准”,评价所建立方法的真实性和可靠性。结果检测血清特异性IgG的灵敏度为100·0%,特异度为97·7%,阳性预测值为97·2%,阴性预测值为100·0%,约登指数为0·977,符合率为98·7%,试验的一致率为98·5%;检测血清中总的特异性Ig灵敏度为97·1%,特异度为95·3%,阳性预测值为94·4%,阴性预测值为97·6%,约登指数为0·925,符合率为96·2%,试验的一致率为97·8%;检测唾液中特异性sIgA的灵敏度为96·2%,特异度为94·5%,阳性预测值为89·3%,阴性预测值为98·1%,约登指数为0·907,符合率为95·1%,试验的一致率为97·5%;检测粪便标本中的特异性sIgA的灵敏度为92·0%,特异度为90·2%,阳性预测值为85·2%,阴性预测值为94·9%,约登指数为0·822,符合率为90·9%,试验的一致率为98·6%,CV值均小于15%。结论该检测方法真实性、可靠性、重复性良好,能满足临床标本检测的要求。     

百日咳黏附素的原核表达及其单克隆抗体的制备

目的原核表达百日咳黏附素(Pertactin,Prn),并制备抗Prn单克隆抗体。方法从无细胞百日咳疫苗生产菌株CS株基因组DNA中克隆Prn基因,插入表达载体pQE-30中,构建重组表达质粒pQE-30-Prn,转化E.coli M15,IPTG诱导表达。表达的重组Prn蛋白经阳、阴离子交换层析纯化后,采用Western blot和ELISA法鉴定重组Prn蛋白的反应原性。以纯化的重组Prn蛋白为免疫原,利用杂交瘤技术制备单克隆抗体,并对制备的单抗进行鉴定。结果重组表达质粒经双酶切和测序鉴定构建正确;表达的重组Prn蛋白相对分子质量约为69 000,主要以包涵体形式表达;纯化的重组Prn蛋白的纯度达95%以上,产量可达25 mg/L,能与不同来源的抗Prn-Ab血清特异性结合。获得2株分泌抗Prn单抗的杂交瘤细胞株,分泌的单抗均为IgG1类,轻链类型均为κ,效价均达1∶105以上,并能特异性识别Prn蛋白,而与百日咳杆菌其他抗原蛋白无交叉反应。结论原核表达、纯化了重组Prn蛋白,并制备了2株效价高、特异性强的单抗,为无细胞百日咳疫苗的质量控制及百日咳杆菌致病机制的研究提供了材料。     

重组单抗药物的非还原电泳纯度分析

目的对重组单抗药物的非还原电泳纯度进行分析,排除样品制备过程中形成的一些假象。方法结合重组单抗的分子结构特征,改变常规SDS-PAGE条件,将抗体样品在不同的缓冲液及电泳条件下进行比较。结果抗体在非还原SDS-PAGE纯度检测中出现的次带,可通过在供试品缓冲液中加入烷基化试剂封闭自由巯基而几乎全部消除;通过降低电泳过程的环境温度,可有效降低主带上方高相对分子质量带的出现。结论单抗药物中由于含有多对二硫键引起的一些因电泳样品制备而形成的假象带,可以通过试验方法的修正而去除。     

Statistical Validation of Reproducibility of HPLC Peptide Mapping for the Identity of an Investigational Drug Compound Based on Principal Component Analysis

Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable “fingerprint” for identity testing and process monitoring. We recently conducted a full method validation study of an optimized reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 monoclonal antibody. We have used this method routinely for over a year to test production lots for clinical trials and to support bioprocess development. One of the difficulties in the validation of the peptide mapping method is the lack of proper quantitative measures of its reproducibility. A reproducibility study may include method and system precision study, ruggedness study, and robustness study. In this paper, we discuss the use of principal component analysis (PCA) to quantitate peptide maps properly using its projected scores on the reduced dimensions. This approach allowed us not only to summarize the reproducibility study properly, but also to use the method as a diagnostic tool to investigate any troubles in the reproducibility validation process.     

Immunological determination of gliadin 33‐mer equivalent peptides in beers as a specific and practical analytical method to assess safety for celiac patients

BACKGROUND: Cereals used for beer manufacturing contain gluten, which is immunotoxic for celiac patients. The gluten remaining after processes of malting and brewing is mostly hydrolyzed, which makes practical evaluation of the immunotoxicity of the gluten pools challenging. RESULTS: We analyzed the presence of gluten peptides equivalent to the major immunotoxic protease‐resistant gliadin 33‐mer in 100 Belgium beers, using monoclonal antibodies (G12/A1). Immunochromatographic strips and enzyme‐linked immonosorbent assay G12/A1 methods estimated at least 20 ppm gluten equivalents in 90 beers and gluten‐free in 10 beers. The G12/A1 reactivity of beer high‐performance liquid chromatographic fractions correlated to the presence of T‐cell‐reactive epitopes identified by peptide sequencing. CONCLUSION: The determination of equivalent gliadin 33‐mer epitopes in beers has been shown to be practical, specific, and sensitive for the measurement of potential immunotoxicity for celiac patients. Copyright © 2012 Society of Chemical Industry     

Simplified method for determining cadmium concentrations in rice foliage and soil by using a biosensor kit with immunochromatography

BACKGROUND: The behavior of cadmium in ecosystems needs to be monitored because of the human toxicity of this heavy metal. The need recently arose for a simple and quick on‐site test for trace levels of Cd in food and environmental samples. In response, an immunochromatographic assay kit for detecting Cd was manufactured by Kansai Electric Power Co. of Japan. This kit uses the antigen–antibody complex reaction between the Cd–EDTA complex and an anti‐Cd–EDTA antibody and shows the results in terms of the degree of color developed on a test paper. We previously reported the successful use of this kit to determine Cd concentrations in brown rice. Here, we applied the kit to the determination of Cd concentrations in rice foliage and soil. RESULTS: Cadmium in rice foliage was not extracted successfully by the method used for brown rice. However, it was successfully extracted by 0.1 mol L^?1 HCl solution at a rice foliage:HCl ratio of 1:20, and coexisting metals were removed sufficiently by the column treatment. The Cd concentrations determined by immunochromatographic assay were well correlated with the values obtained by acid decomposition and inductively coupled plasma mass spectrometry. The 0.1 mol L^?1 HCl‐extractable Cd concentration in soil was also determined successfully with the kit. CONCLUSION: Approximate Cd concentrations in rice plants and 0.1 mol L^?1 HCl‐extractable Cd concentrations in soil can be monitored easily and quickly by this method at locations where facilities for acid digestion and precision analysis are not available. Copyright © 2009 Society of Chemical Industry     

Detection of human serum albumin on protein array using scanning tunneling microscopy

Electrical detection of biological binding events, such as protein–protein interaction and DNA hybridization, has emerged as an alternative method to conventional colorimetric and fluorescence based methods. In this study, we demonstrate an electrical detection technique of protein array which can be simply extended for multifunctional measurements and detection of biological binding events on micro-scale array. Micro-contact technique was used for the fabrication of protein chip. The fabricated protein array on Au substrate was characterized by fluorescence microscopy and scanning tunneling microscopy (STM). The chip was designed to investigate immunocomplexes comprised of our model protein, human serum albumin (HSA), corresponding antibody fragments, and Au nanoparticle–antibody conjugates. The peak-like pulse obtained by electrical tunneling current between these complexes and the STM tip varies on the surface density of the bound complexes. Using the electrical detection technique based on STM, 100 fg/mL of HSA could be successfully detected by STM. Importantly, the proposed concept of measurement could allow multiple analyses of analytes at nano-scale array, which is difficult to be analyzed by conventional fluorescence based method.     

电离辐射与淋巴细胞

早期的临床工作是了解淋巴细胞的辐射剂量与效应关系,作为初期最敏感的指标之一。对原子弹受害者的淋巴细胞也进行了远期效应的观察。实验室工作方面研究了淋巴细胞的活力、死亡率,玫瑰花结、细胞膜和DNA等分子的变化。染色体畸变,微核率及其相互关系的研讨也引起人们重视。 应用PHA、ConA、PWM、LPS分别激活淋巴细胞的各个亚群,研究其辐射效应。有关PHA激活的T淋巴细胞的辐射效应研究得较多。作者察觉PWM和ConA激活的淋巴细胞对辐射敏感,均减弱了辅助B淋巴细胞转化的作用,PHA激活的淋巴细胞受影响较轻,LPS激活的淋巴细胞变化最小。 单克隆抗体技术研究淋巴细胞亚群的进展为进一步研究淋巴细胞的辐射效应提供条件,可以了解辐射影响免疫功能的主要环节。 有的作者研究了各种病人的淋巴细胞的辐射敏感性,以了解免疫方面存在的缺陷,还可作为制定辐射治疗某些白血病方案的依据。