Natural infection with raspberry bushy dwarf virus (RBDV) of the putatively RBDV-resistant red raspberry cultivar Glen Moy, and the demonstration that it does not contain the RBDV resistance gene, Bu

When released to commerce in 1981, the red raspberry cv. Glen Moy was reported to be immune to the Scottish type isolate of raspberry bushy dwarf virus (RBDV-D200). Field observations of this cultivar in localities where RBDV was prevalent tended to support this claim of its resistance, but in the past 6–10 yr, RBDV infection has been reported in this cultivar in Australasia, USA and in several commercial crops in England. Therefore, experiments were made to investigate the reason(s) for this apparent anomaly using RBDV-infected material, putatively of cv. Glen Moy, from two locations in southern England and one each from Australia, New Zealand (NZ) and the USA. Genetic fingerprinting of genomic DNA from samples of these five RBDV-infected raspberry sources confirmed their identity as cv. Glen Moy. Comparisons of some serological and genomic properties of the five Glen Moy RBDV isolates indicated that, whilst they shared many properties with previously well characterised isolates of this virus, they were distinguishable from them. Characterisation of the isolate from NZ maintained in raspberry showed that it did not have a Rubus host range characteristic of resistance-breaking (RB) isolates, indicating that for this location, and probably also for those of Australia and the USA, RB isolates were not the cause of infection in cv. Glen Moy. When virus-tested plants of cv. Glen Moy and 45 progeny seedlings from the cross between cv. Glen Moy and the RBDV-susceptible cv. Autumn Bliss were graft inoculated with RBDV-D200, all grafted plants became infected indicating that cv. Glen Moy does not contain the RBDV resistance gene, Bu. Possible reasons for the previously reported resistance of cv. Glen Moy to RBDV are discussed.     

Recognition sites of monoclonal antibodies A4 and A24 in the α-latrotoxin molecule

α-Latrotoxin (α-LTX) binding sites to functionally active monoclonal antibodies (MA) A4 and A24 were localized using three approaches: hydrolysis of the toxin followed by theN-terminal sequencing of immunoreactive peptides; the study of antibody interaction with several recombinant α-LTX fragments; Western immunoblotting of synthetic overlapping peptides (6–8 aa) whose structures correspond to that of the immunoreactive α-LTX fragment. It was shown that the MA A4 epitope is located within the F²³⁴-M²⁹⁴ protein fragment and that MA A24 interacts with the fragment³⁴⁷FDKDIT³⁵².     

Recognition of Superpotent Sweetener Ligands by a Library of Monoclonal Antibodies

Monoclonal antibodies (mAb) made to the superpotent guanidino sweet tasting ligand, N-(p-cyanophenyl)-N-(diphenylmethyl)-guanidineacetic acid were examined for their molecular recognition specificities using 14 different sweetener analogues in a competitive radioimmunoassay. The effects of variations in pH on ligand binding was also examined by radioimmunoassay. Photoaffinity labelling of the binding site was accomplished using a radiolabelled azido-derivative of the parent ligand, and L-chain or H-chain labelling was easily identified in several different mAb. For two of the mAb examined in this study (NC6.8 and NC10.14), the analogue binding studies are in agreement with the known Fab-ligand crystal structures. Monoclonal antibodies to this family of sweet tasting compounds may be useful probes for the study of sweet taste chemistry and identification of novel sweet taste ligands from combinatorial chemical libraries. © 1997 John Wiley & Sons, Ltd.     

Hybridomas: A new dimension in biological analyses

Summary Since the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic increase in publications using these reagents has been in itself fascinating and informative. An overview of the development of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules, differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the regulation of growth control and cell division are provided. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association. Washington, D.C., June 7–11. 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Selection of resistance breaking strains of tomato spotted wilt tospovirus

In glasshouse tests, sap from plants infected with 15 different isolates of tomato spotted wilt tospovirus (TSWV) from three Australian states was inoculated to nine genotypes of tomato carrying TSWV resistance gene Sw-5 or one of its alleles. A further two resistant tomato genotypes were inoculated with four isolates each. The normal response in resistant genotypes was development of necrotic local lesions in inoculated leaves without systemic invasion, but 22/752 plants also developed systemic reactions in addition to local hypersensitive ones. Using cultures from two of these systemically infected plants and following four cycles of subculture in TSWV resistant tomato plants, two isolates were obtained that gave susceptible type systemic reactions but no necrotic spots in inoculated leaves of resistant tomatoes. When these two isolates, Da_WA-1d and To_TAS-1d, were maintained by repeated subculture for 10 successive cycles in Nicotiana glutinosa or a susceptible tomato genotype, they still induced susceptible type systemic reactions when inoculated to resistant tomato plants. They were therefore stable resistance breaking isolates as regards overcoming gene Sw-5. When resistance-breaking isolate Da_WA^-1ld multiplied together with original isolate Da_WA-l in susceptible tomato, it was fully competitive with the original isolate. However, when Da_WA-ld and To_TAS-ld were inoculated to TSWV resistant Lycopersicon peruvianum lines PI 128660R and PI 128660S and to TSWV resistant Capsicum chinense lines PI 152225, PI 159236 and AVRDC CO0943, they failed to overcome the resistance, producing only necrotic local lesions without systemic infection. Thus, although the ease of selection, stability and competitive ability of resistance breaking isolates of TSWV is cause for concern, L. peruvianum and C. chinense lines are available which are effective against them. The effectiveness of the resistance to TSWV in nine tomato genotypes was examined in a field experiment. Spread was substantial in the susceptible control genotype infecting 42% of plants. Resistance was ineffective in cv. Bronze Rebel, 26% of plants developing infection. In contrast, it held up well in the other eight resistant genotypes with only 1–3 or no plants of each becoming infected. Accumulated numbers of Thrips tabaci, Frankliniella occidentalis and F. schultzei were closely correlated with TSWV spread.     

人红细胞生成素受体胞外区克隆及序列测定

人红细胞生成素受体 (h EPOR)是人红细胞生成素的作用配体 ,其胞外区是 h EPO的作用域 ,它的克隆、表达对两种分子的相互作用机制以及 EPO类似物 (新型造血药物 )的筛选都有十分重要的意义。以人胎肝为材料 ,通过对其总 RNA的提取 ,利用 RT- PCR方法扩增 h EPOR的胞外区基因和跨膜区基因及推导相应的氨基酸残基排列 ,结果与国外文献报道相比较从而检验其正确性。     

Antigenic components of human glioblastoma multiforme and rat C6 glioma using monoclonal antibodies

With whole U87MG cells used as antigenic stimulant, two clones 1A5G6 and 1D3A3 secreted monoclonal antibodies which gave intense staining in monolayer cultures of the cells as ascertained by indirect immunofluorescence. Antibodies from clone 1A5G6 stained both the cytoplasm and the processes, and that from clone 1D3A3 stained only the cytoplasm and not the processes. 1A5G6 elicited no cross-reactivity towards human fetal and adult brain and lungs, liver, kidney or spleen, mouse neuroblastoma and melanoma, rat C₆ glioma, neuroblastoma X glioma hybrid and normal rat kidney cells. It gave 58–60% cross reactivity with the human neuroblastoma and T-cell leukemia cells. The antigenic comPonent has been identified to be a membrane protein of molecular weight 25–30 kilodaltons by immunoblotting. Using C₆ glioma cells as antigenic stimulant 19 clones which were positive for C₆ glioma cells, but negative for rat liver cells as inferred by indirect immunofluorescence were selected. Antibodies secreted by all these gave positive reaction towards normal rat kidney and fetal rat kidney cells in culture. Distinct identity of these clones were ascertained by discernible staining patterns in indirect immunofluorescence on C₆ glioma cells.     

Changes in the Expression of Membrane Antigens During the Differentiation of Chicken Erythroblasts

Chicken erythroblasts can be transformed by the avian retrovirus, avian erythroblastosis virus (AEV). Earlier studies have shown that the mechanism of transformation appears to involve a “block” in differentiation, in that when erythroblasts are transformed by a temperature-sensitive mutant of ts34 AEV and incubated at the nonpermissive temperature, the cells start to differentiate and produce hemoglobin. We have decided to use this system to isolate pure populations of chicken erythroblasts and raise monoclonal antibodies against their cell surface proteins. Three monoclonal antibodies were isolated and tested for their ability to bind to various hematopoietic cell types; two were shown to be erythroid-specific, whereas the other antibody bound to proliferating cells but not to erythrocytes or granulocytes. Of the erythroid-specific antibodies, one precipitated a 94,000 molecular weight protein, whereas the other precipitated a 11,000 molecular weight protein that was tentatively identified as hemoglobin. The use of this system and approach to identify and evaluate changes that occur during the differentiation is discussed.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

Summary We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, resuable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Mouse monoclonal antibodies specific for endothelial cells

Summary Balb/c mice were immunized with a human endothelial cell pool. Spleen cells were then fused with a NS-0 hybridoma cell line. A number of hybridomas secreted antibodies that reacted with the immunizing endothelial cell pool as well as with every other tested umbilical cord vein~derived human endothelial cell. These monoclonal antibodies also stained pig, rabbit and ox aortic endothelial cells indicating their specificity for this cell type. Five of 16 monoclonal antibodies additionally reacted with human fibroblasts (HFIB). The produced monoclonal antibodies did not recognize FVIIIRAG or MHC determinants. They can therefore be regarded as additional and reliable markers for endothelial cells in vitro.     

Cardiac myofibrillar proteins: Biochemical markers to estimate myocardial injury

Ischaemic heart disease represents the most common of the serious health problems in the contemporary society and acute myocardial infarction (AMI) is the major cause of cardiovascular morbidity and death. The accurate localization and determination of the infarct size and the volume of myocardium at risk at the time of insult is crucial and vital for the choice of treatment. Initially the ischaemic cells are reversibly injured. However, if these changes are not reverted at the earliest, it results in the death of the myocyte. This irreversible myocyte necrosis travels transmurally towards epicardium in the form of a wavefront [1]. A timely intervention during evolving infarct could reduce and delimit the infarct and preserve the left ventricular function [2].Enzyme analysis and electrocardiography (ECG) along with the clinical history of the patient is still considered to constitute a reliable triad in the diagnosis of myocardial infarction (MI) [3]. Efforts have been made to relate infarct size with the serum enzyme level changes without much success. In addition, a number of specialist techniques such as planar radioisotope imaging, single photon emission computed tomography (SPECT), positron emission tomography (PET), Echocardiography, Ventriculography and nuclear magnetic resonance (NMR) imaging have been devised to support diagnosis in the patients who show ambiguous symptoms and ECG findings. However most of these procedures are unavailable to the patients due to economic reasons while others have suffered due to non-availability of ideal radiopharmaceuticals. Major advances have been made in the methods based on immunological techniques to improve the detection and estimation of infarct. These methods are exclusively based upon the production and availability of specific antibodies against intracellular, cardiac specific components [4].     

Proteins of the acrosomal region in mouse sperm: Immunological probes reveal post-testicular modifications

Effects of reduced glutathione (GSH), oxidized glutathione (GSSG), or glutathione reductasc (GR) supply were studied on the ability of hamster oocytes to be fertilized by human sperm. Zona-free oocytes were pretreated with these compounds prior to sperm insemination. Oocyte pretreatment with high concentrations of GSH or GSSG (50 or 100 mM. 30 min) significantly increased the penetrated oocyte rate (PR). Polyspermy was not increased except when high concentrations of GSH (100 mM) were used. Incubation of oocytes with GR (1 or 10IU/ml) prior to sperm insemination induced increasing dose-dependent PR. Polyspermy increased significantly with 10 mM GR in oocyte incubation medium. Oocyte incubation for 30 min with the sulfhydryl blocking agent iodoacetamide (1 mM) led to a drastic decrease in oocyte penetration and in polyspermy. Our results demonstrate an original way to increase the efficacy of human-hamster heterospecific fertilization. Various hypotheses are discussed explaining these observations which open new investigations for heterospecific and homospecific in vitro fertilization.     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Monoclonal antibodies – a proven and rapidly expanding therapeutic modality for human diseases

The study of antibodies has been a focal point in modern biology and medicine since the early 1900s. However, progress in therapeutic antibody development was slow and intermittent until recently. The first antibody therapy, murine-derived murononab OKT3 for acute organ rejection, was approved by the US Food and Drug Administration (FDA) in 1986, more than a decade after César Milstein and Georges Köhler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975. As a result of the scientific, technological, and clinical breakthroughs in the 1980s and 1990s, the pace of therapeutic antibody discovery and development accelerated. Antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development. Despite the progress, need for improvement exists at every level. Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.