Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.     

人体外形的相似性

本文首先提出了人体相似性的概念和定义, 并进行了数学论证, 导出了标准人体的数学表达方法,然后, 根据90名志愿者的样本具体计算说明了标准人体尺寸的适用性。本文提出的标准人体尺寸处理方法, 可供人种和体型分类、人体模型与服装设计时确定人体各部尺寸, 为利用人体其他节段估测人全身尺寸的可能性提供参考。最后指出, 表示人体尺寸间相互关系的人体常数是存在的; 在设计人体模型时, 不管模型的比例如何, 所设计模型的人体常数需保持与所代表的人群样本的人体常数一致。     

基于Hartmann—Shack传感器的人眼波前像差重建技术

阐述了人眼波前像差的概念及其Zernike多项式表示方法,着重讨论了Hartmann—Shack传感器测量人眼像差的机理和人眼波前像差重建的方法,并对人眼波前像差重建系统进行了详细设计。该技术给人眼像差的校正以及角膜“个体化切削”的实现带来了新契机。     

Use of human germline genes in a CDR homology-based approach to antibody humanization

We report a new method of humanizing antibodies by complementarity determining region (CDR) grafting. Our method differs from others in that we choose human framework sequences from the set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. The structural similarity is evaluated by scoring residue-to-residue homology of the mouse CDRs to human candidates with the same Chothia canonical structures. The method is illustrated with the humanization of the anti-lysozyme antibody D1.3.     

Combined LC/MS-platform for analysis of all major stratum corneum lipids,and the profiling of skin substitutes

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10 μl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, ‘high-throughput’ profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.     

A structured hazard identification method of human error for shale gas fracturing operation

Shale gas fracturing is a complex system of continuous operation. If human errors occur, it will cause a chain reaction, from abnormal events to fracturing accidents, and even lead to abandonment of shale gas wells. The process of shale gas fracturing has many production stages that are complex and the consequence of any error is serious. The human error modes in shale gas fracturing process are mutative. Therefore, human error should be studied in a systematic way, and in a hybrid framework, that is, whole integration of identification, prioritization, reasoning, and control. A new structured identification method of human error in a hybrid framework for shale gas fracturing operation is presented in the article. First, human error is structurally identified based on the human HAZOP method. Second, fuzzy VIKOR method is applied to comprehensive prioritization. Finally, 4M element theory is used to analyze the human error and control its evolution. The method improves the consistency of the identification results through the standard identification step and the identification criterion. Results from a study of feed-flow process indicate that 34 kinds of human errors can be identified, and high-probability errors occur in the behavior of implementation and observation.     

Genogeography of human populations: computer mapping of population genetics data

Genogeography as a field of interdisciplinar investigation was introduced into science in 1928 by Russian geneticist A. S. Serebrovsky an today is well known in human genetics as gene geography. This work was undertaken to introduce a new method of computer cartography of gene frequencies in human and in any other populations. The method is different from known one of P. Menozzi, A. Piazza and L. Cavalli-Sforza. Two key moments of the method are principle of fusion-fission of gene in the homogeneous geographical space equally free-for-all human genes, and principle of local-linear (but not of the high-orders) interpolation of gene frequencies onto spheric surface of geographical space. Such procedure was used for mapping of human AB0-B gene frequencies among native populations of Central Asia.     

Secondary structure prediction of anterior pituitary hormones. Lack of correlation between predicted values and circular dichroism data.

The secondary structures of human somatotropin, human choriomammotropin, ovine and porcine prolactin, human, ovine and porcine beta-lipotropin, human and ovine lutropin, human thyrotropin, human corticotropin, alpha-melanotropin and human beta-melanotropin have been predicted by the method of Chou & Fasman. Predicted contents of alpha-helix and beta-sheet do not correspond well with values estimated from circular dichroism spectra.     

Isolation and characterization of human DNA in agarose block

Classic phenol-chloroform extraction of human DNA results in substantial shearing and low yield. Because DNA analysis in human genetic disorders needs relatively intact DNA, we modified the existing method and systematically analyzed the human DNA isolated from HeLa cells, leukocytes, amniocytes, and fibroblasts in agarose block and compared the results to those obtained by conventional phenol method. Our results showed that DNA isolated by the agarose method was higher in molecular weight, with minimal shearing as compared to the phenol method. Yield of DNA from the agarose method was substantially higher, almost twice that obtained by the phenol method. Restriction enzyme digestion of DNA from the agarose method indicated the usefulness of this DNA for restriction fragment length polymorphism (RFLP) analysis without further purification. DNA obtained by the agarose method was found to be more resistant to thermal degradation and more stable on long-term storage than that of phenol-extracted DNA.     

Radioimmunoassay of low molecular weight human kininogen

A radioimmunoassay for low molecular weight (LMW) human Kininogen has been carried out. The first step was to prepare LMW Kininogen from human plasma. The proposed method allowed to get chemically pure and biologically active LMW Kininogen. This preparation was used to induce antibody. Optimal conditions for labelling and incubation were determined. This method may be applied to the assay of Kininogen in human plasma.     

Rapid estimation of binding activity of influenza virus hemagglutinin to human and avian receptors

A critical step for avian influenza viruses to infect human hosts and cause epidemics or pandemics is acquisition of the ability of the viral hemagglutinin (HA) to bind to human receptors. However, current global influenza surveillance does not monitor HA binding specificity due to a lack of rapid and reliable assays. Here we report a computational method that uses an effective scoring function to quantify HA-receptor binding activities with high accuracy and speed. Application of this method reveals receptor specificity changes and its temporal relationship with antigenicity changes during the evolution of human H3N2 viruses. The method predicts that two amino acid differences at 222 and 225 between HAs of A/Fujian/411/02 and A/Panama/2007/99 viruses account for their differences in binding to both avian and human receptors; this prediction was verified experimentally. The new computational method could provide an urgently needed tool for rapid and large-scale analysis of HA receptor specificities for global influenza surveillance.     

蒙特卡罗方法和三维数字人体模型在放疗计划质量保证中的应用

放射治疗的质量保证是保证放射治疗成功的有力方法。对于放疗计划的验证和评估有CT模拟机、仿体等方法,这些方法各有优缺点。文章提出了一种用人体图像数据构造仿真模型的方法,并用蒙特卡罗软件和美国“可视人项目”的数据集计算该模型在接受放射治疗时体内剂量的三维分布。由于采用人体的真实图像数据,以及蒙特卡罗方法计算粒子输运时的准确性,该方法能够得到真实的三维剂量分布。     

人B细胞永生化研究进展

人源单克隆抗体具有免疫原性低、半衰期长等优势,成为了体内应用中不可或缺的生物制剂.人类抗体库为人源单克隆抗体的制备提供了丰富的来源,人B细胞永生化是获得人类抗体库的潜在有效方法,可应用于人源单克隆抗体的制备.由于各平台均有亟待解决的问题,基于人B细胞永生化的抗体制备尚局限在实验室研究阶段,且目前尚缺乏一篇系统综述以明确...     

The disappearance rate of human versus rat intermediate density lipoproteins from rat liver perfusion.

The aim of this work was to compare the disappearance rate of human and rat intermediate density lipoproteins (IDL) using the rat liver perfusion system. Human and rat IDL were produced in vitro by incubating human or rat very low density lipoproteins (VLDL) with either rat post-heparin plasma (method I) or a resolubilized isopropanol precipitate of rat post-heparin plasma (method II). With both methods, the degree of triacylglycerol lipolysis was approximately 55%. The different preparations of IDL were labelled with 125I and added to perfusates of rat livers. The disappearance rates of 125I-labelled IDL were monitored by measuring the radioactivity associated with apolipoprotein (apo) B in the perfusate during a 15-min period. Both human and rat IDL prepared with method I had an increased apoE to apoC ratio as compared with their native counterparts. Furthermore, human IDL had a significantly higher apoE to apoC ratio than rat IDL. However, when IDL were produced in the absence of exchangeable apolipoproteins (method II), no change in the apoE to apoC ratios was observed for the transformation of VLDL to IDL and the ratios were similar for human and rat IDL. Despite these differences, human IDL were always removed at a lower rate than rat IDL. The only striking difference between the two types of IDL made by method II was that the apoB100 to apoB48 ratio was considerably higher in human than in rat IDL. These results suggest that the apoB100 to apoB48 ratio is likely to be responsible for the observed differences in liver uptake between rat and human IDL.     

Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection

A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (%C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma.     

In vitro immunization of human peripheral blood lymphocytes: establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method. This revised version was published online in August 2006 with corrections to the Cover Date.     

不同干扰程度下土壤有机质空间最优插值法研究

选择最优空间插值方法对明确不同干扰程度下干旱区土壤有机质空间分布规律具有重要意义。通过对新疆阜康市土壤采样分析,分别运用普通克里格法(Ordinary Kriging, OK)、反距离权重法(inverse distance weight, IDW)、径向基函数法(radial basis function, RBF)、局部多项式法(local polynomial, LPI)4种插值方法,描述不同人为干扰程度下干旱区土壤有机质空间分布特征,以探讨其最优空间插值方法。结果表明:①随干扰程度增加,土壤有机质含量降低,空间变异性由弱到中等,变异系数由9.27%增加至28.7%;同时,土壤有机质空间自相关性逐渐降低,从无人为干扰区、人为干扰区到重度人为干扰区分别呈强烈、中等、弱相关关系,即干扰程度越强受随机因素作用越大。②虽然4种方法的插值精度均随干扰程度的增强而降低。但OK法在空间结构性较强的无人为干扰区插值效果最好,R~2为0.625;在人为干扰区及重度人为干扰区RBF法的插值精度均最高,R~2分别为0.562和0.434。该结果为寻找适合于不同干扰程度下干旱区土壤有机质的空间插值方法提供一定的科学参考。     

High-performance liquid chromatographic method for the determination of gabapentin in human plasma

A sensitive high-performance liquid chromatography (HPLC) method using UV detection for the determination of gabapentin in human plasma has been developed. In this method, gabapentin was extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge followed by derivatization with phenylisothiocyanate. Analysis was achieved by using a HPLC system that was equipped with a UV detector. The quantitation limit of gabapentin in human plasma was 0.03 microg/ml. The method is sensitive with excellent selectivity and reproducibility and it has been applied to a bioequivalence clinical study with great success.     

Synteny comparison between apes and human using fine-mapping of the genome

Comparing the genomes of the great apes and human should provide novel information concerning the origins of humankind. Relative to the great apes, the human karyotype has one fewer chromosome pair, as human chromosome 2 derived from the telomeric fusion of two ancestral primate chromosomes. To identify the genomic rearrangements that accompanied human speciation, we initiated a comparative study between human, chimpanzee, and gorilla. Using the HAPPY mapping method, an acellular adaptation of the radiation hybrid method, we mapped a few hundred markers on the human, chimpanzee, and gorilla genomes. This allowed us to identify several chromosome rearrangements, in particular a pericentric inversion and a translocation. We precisely localized the synteny breakpoint that led to the formation of human chromosome 2. This breakpoint was confirmed by FISH mapping.     

Increased production of low molecular weight recombinant proteins in Escherichia coli.

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-Xaa-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.