Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

Differential toxinogenesis in the genusPichia detected by an anti-yeast killer toxin monoclonal antibody

The differential toxinogenesis of 25 isolates belonging to species of the potential yeast killer genusPichia that were previously classified in the genusHansenula was comparatively demonstrated by two serologic techniques (indirect immunofluorescence and double immunodiffusion) by using a monoclonal antibody against a yeast killer toxin produced by a selected strain ofPichia anomala (UCSC 25F). The killer phenotypes of thePichia isolates were evaluated by their ability to kill each other. The results, although of insufficient taxonomic value for a reliable separation of either species or genera, attest to the genomic heterogeneity for the killer character in the genusPichia as well as the presumptive dual killer/sensitive identity for each single isolate.     

Isolation and characterization of monoclonal antibodies reactive with endothelial cells

Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function.     

An immobilized hybridoma culture perfusion system for production of monoclonal antibodies

A fixed bed perfusion system for hybridoma cell immobilization is presented. The system consists of a culturing vessel (300 ml total volume) in which polyurethane (PU) sponges in the form of small cubes of about 5 mm sides are packed. Cells are immobilized by physical entrapment in the foam matrix. By entrapment of the cells in the pores of the matrix high cell concentration can be maintained in a mechanically protected environment. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber (700 ml total volume).Medium flow rate, feeding rate, dissolved oxygen, pH, nutrient uptake and waste product formation can be easily monitored and controlled. Steady state conditions are established with medium dilution rates of 1.0–1.5 reactor volume per day. The steady state is characterized by a constant cell density, constant culture volume and constant glucose and lactate levels. Cell-free supernatant is collected continuously in a cold room adjacent to the 37°C culture room. Monoclonal antibodies (MAb) are produced at a concentration of 150–200 g/ml for several weeks. An important feature of the system is the capacity to maintain a population of cells after the growth phase in a non-proliferating state for extended time periods expressing high titers of MAb.Abbreviations DO Dissolved Oxygen - FBS Fetal Bovine Serum - FBR Fixed Bed Reactor - MAb Monoclonal Antibody - PU polyurethane     

Immunological approach to characterize proacrosin and various acrosin forms in boar and man by monoclonal antibodies

Three different monoclonal rat antibodies, Acr1, Acr2, and Acr3, have been established against boar proacrosin. They are shown by enzyme-linked immunosorbent and immunoblot assays to react with boar proacrosin and several different acrosin molecules derived therefrom during activation. The epitopes detected by the three antibodies are different from each other, one being highly sensitive to reduction and periodate treatment. The antibodies crossreact with various proacrosin and acrosin molecules derived from human sperm extract; they also show indirect immunofluorescent staining of the acrosomal region of ejaculated sperm from normal men but fail to react with round-headed spermatozoa.     

Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation

A novel and powerful fermentation method is reported for the large-scale growth of mammalian cells and their secreted products. The system described illustrates many of the advantages of conventional batch fermentation processes but in addition has been shown to yield cell densities in excess of 1×10⁷ cells/ml with concomitant increase in product concentration.Abbreviations MAb Monoclonal Antibody - STR Stirred Tank Reactor - FBS Foetal Bovine Serum     

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.     

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.     

CK-MB特异性单抗的制备方法及其应用

本研究拟建立肌酸激酶同工酶MB(CK-MB)特异性单克隆抗体(m Ab)的研制方法,对抗CK-MB单抗进行评价分类及性质鉴定,并初步建立CK-MB定量检测试剂。以CK-MB抗原免疫BALB/c小鼠,利用常规单抗制备技术,使用间接和捕获ELISA差异筛选法筛选单抗。利用肌酸激酶同工酶(CK-MM/BB/MB)抗原对所制备单抗的抗原识别表位进行鉴定,另通过免疫印迹法及合成CK-MM、CK-BB差异性的线性表位肽鉴定对所制备的单抗进行评价分类。使用双抗体夹心ELISA方法筛选检测CK-MB抗原的配对m Ab,并初步建立CK-MB定量检测试剂。使用74例临床标本初步评价该试剂与罗氏试剂的检测一致性。最终,我们成功筛选到22株稳定分泌抗CK-MB抗体的杂交瘤细胞株,这些单抗可以分为线性、偏构象的CK-MB和CK-MM或者CK-BB交叉的单抗以及与CK-MB特异反应的偏构象型单抗,并使用偏构象型单抗研制出CK-MB定量检测试剂,该试剂与罗氏试剂相关系数r达到0.930 9。综上所述,本研究建立了研制CK-MB偏构象型特异性单抗的筛选方法,通过对所筛选的单抗进行分析鉴定并建立了CK-MB定量检测试剂,与罗氏试剂检测结果符合率高。     

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns

Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation‐associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The use of killer sensitivity patterns for biotyping yeast strains: the state of the art, potentialities and limitations

In recent years molecular techniques have been the most useful tools for the unequivocal identification of undetermined strains at the species level. In many instances, however, a further discrimination at the strain level (biotyping) is required, such as during epidemiological investigations, in which the distribution of pathogenic microorganisms is studied, and for patent protection purposes. Although molecular methods are routinely used also for yeast biotyping, several nonmolecular techniques have been proposed. One of these, the determination of the killer sensitivity pattern (KSP) towards a panel of selected killer toxins has proven to be a good auxiliary method. Despite the plethora of studies published, the potential and limitations of the determination of KSPs have never been critically evaluated. In this review the use of this nonmolecular technique as a biotyping tool is discussed and compared with some currently used DNA-based procedures. In addition, methodological, mechanistic and ecological implications are evaluated.     

Immunofluorescent staining method for use with monoclonal antibodies

This note describes an immunofluorescent staining method for cells in the S-phase which have been allowed to take up bromodeoxyuridine into their DNA in place of thymine. The technique involves the use of fluorescinated monoclonal antibodies against bromodeoxyuridine and allows rapid and accurate estimation of cells in the S-phase, the technique does not require interpretation by skilled technicians.     

Characterization of carp thrombocytes with specific monoclonal antibodies

Monoclonal antibodies (WCL6) specific for carp Cyprinus carpio thrombocytes were produced by immunizing mice with membrane lysates of IgM-negative peripheral blood leucocytes (PBL) and selected on the negative reaction with B cells. WCL6 was reactive with a membrane molecule of approximately 90 kDa and to a lesser extent with molecules of approximately 95 and 110 kDa. In general, between 30 and 40% of PBL were WCL6⁺ and appeared to be round to spindle-shaped cells. Immunohistochemical analysis of cryo-sections showed much higher numbers of WCL6⁺ cells in the spleen than in the pronephros, intestine and thymus. Flow cytometric analysis of cell suspensions isolated from these organs only revealed WCL6⁺ cells (4–10%) in the spleen. Electron microscopy of immunogold-stained WCL6⁺ PBL showed round but also some spindle-shaped cells with canalicular and granular structures, and a more irregular and electron-dense nucleus than found in lymphocytes. WCL6⁺ cells with electrondense pyknotic nuclei (without a clear nuclear envelope) were found also and their frequency increased with the length of the isolation and staining procedure used. In the spleen, several differentiation steps of WCL6⁺ cells were found and hence the spleen seems to be the thrombopoietic organ in carp. Thrombocytes from blood could be activated with collagen; the collagen-activated cells showed a higher side (90°) scatter by flow cytometric analysis and finally considerable cell death.     

Specific detection of the Andean strain of potato virus S monoclonal antibodies

Four mouse monoclonal antibodies (MAbs) specific for the Andean strain of potato virus S (PVS^A) were produced. The MAbs reacted with four isolates of PVS^Abut did not react with four isolates of ordinary strain of PVS (PVS^O). The MAbs did not react with six other members of the Carlavirus group including potato virus M. A MAb-based ELISA, using MAbs (IEB-1 and IEB-4-AP), was devised and shown to specifically detect PVS^A.     

Linking radiosilver to monoclonal antibodies reduced by ascorbic acia

Radiosilver-111 and Radiogold-199 were proposed by us (1) as suitable isotopes for radioimmunotherapy in areas such as India by reason of their suitable half-lives and B-emissions (Ag-111T _1/2=7.45 d and Au-199T _1/2=3.15 d). Since silver is monovalent, it is difficult to link to conventional bifunctional chelates. We therefore explored the use of sulfur-based linkers (2). Encouraged by the Thakur and De Fulvio Technique (3) of linking technetium to disulfide groups in antibodies reduced by ascorbic acid that is eminently biocompatible, we have explored the linkage of silver to immunoglobulin reduced by ascorbic acid. The linkage of silver was assessed with stable Ag-108 using dialysis to quantify the free silver after the reaction of silver and reduced immunoglobulins in various molar ratios (1∶1, 1∶2, 1∶5, 1∶10). The silver quantity was estimated gravimetrically after precipitation as chloride. It was observed that using these molar ratios there was negligible silver efflux into the dialysate, suggesting stable linkage. We also assessed the linkage using Ag-110M as radiotracer. The comparative results with the two techniques are described.     

Stimulation of intestinal Na+/d-glucose cotransport by monoclonal antibodies

Summary The small intestinal brush border membrane is endowed with a number of transport systems. Monoclonal antibodies were produced against integral membrane proteins and tested for their ability to bind to such membranes. For this purpose papain-digested, deoxycholate-extracted BBMVs from rabbit small intestine were used to immunize mice. Of the 765 hybridoma supernatants tested, 119 gave a significantly higher extent of binding to the crude antigen preparation as compared with the background. The monoclonal antibodies were also tested for their ability to influence the sodium-dependent uptake of solutes into intact BBMVs. Two monoclonal antibodies clearly showed stimulation of secondary actived-glucose transport, whereas sodium-dependent uptake ofl-alanine andl-proline was not affected. Hydrophobically labeled, i.e. intrinsic, membrane proteins of 175, 78 and 65 kilodaltons could be immunoprecipitated by both monoclonal antibodies, the 78 kDa band corresponding in all likelihood to the Na⁺/glucose cotransporter.     

白背飞虱单克隆抗体的制备及其特性的研究

应用杂交瘤技术,制备出4株高度特异性的白背飞虱单克隆抗体,分别命名为WPH-1H9、WPH-2B6、WPH-2E12和WPH-3F12。这些抗体与其它8种昆虫未发生交叉反应,其中WPH-2B6可与白背飞虱所有虫态发生反应,其余3株只与卵和雌成虫发生反应。应用免疫双扩散法鉴定抗体类型及亚类,结果表明：WPH-2B6为IgG_2b亚类,其余均为IgG₁亚类。SDS-聚丙烯酰胺凝胶电泳和Western blot印迹分析表明,白背飞虱抗原主要由分子量分别为182、116、66.2及40 kD的4个多肽组成,其中WPH-2B6与182、116 kD的多肽结合,其余3株的单抗只与116 kD的多肽具有亲和性。最后对这些单克隆抗体在捕食作用研究中的应用潜力进行了讨论。     

Production of monoclonal antibodies to the prion protein and their characterization

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25–36 of PrP corresponds to one antibody, and epitopes located in region 222–229, to the other two. The antibodies to fragment 222–229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.     

Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption

To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose-based matrix (Stream-line-SP), and a self-derivatized material based on controlled-pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose-based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. (c) 1995 John Wiley & Sons, Inc.     

Preparation and characterization of monoclonal antibodies to human hexokinase type I

1. Three different immunization protocols and several screening procedures were used to prepare seven mouse monoclonal antibodies to human placenta hexokinase type I. None of these monoclonals were able to recognize the native enzyme but all detected hexokinase when adsorbed onto polystyrene plates or on immunoblots after SDS/polyacrylamide-gel electrophoresis. 2. All seven monoclonals recognize the two different subtypes of human hexokinase I equally well. Limited tryptic digestion of hexokinase followed by Western blotting and immunodetection show that these monoclonals recognize epitopes that lie in different tryptic peptides. 3. Comparative ELISA studies showed that human hexokinase types I and II have great immunological similarities while hexokinase I from different mammalian species and yeast hexokinase are recognized with different affinities.