Antibody-producing Hybridomas Research Articles

The CCL2-CCR2 axis determines whether glomerular endocapillary hypercellularity or wire-loop lesions develop through glomerular macrophage and neutrophil infiltration in lupus nephritis.

The CCL2-CCR2 axis is involved in lupus nephritis, however the precise roles in the mechanisms by which different pathological lesions develop after glomerular immune complex deposition remain elusive. Previously, we demonstrated that genetic CCR2 inhibition induced a histological switch from glomerular endocapillary hypercellularity to wire-loop lesions in murine lupus nephritis. This study aimed to clarify the CCL2-CCR2 axis-mediated cellular mechanism in the formation of these different pathological lesions. We injected MRL/lpr mouse-derived monoclonal IgG3 antibody-producing hybridomas, 2B11.3 or B1, into wild-type (WT) mice to selectively induce glomerular endocapillary hypercellularity or wire-loop lesions. The expression of chemokine and chemokine receptors was analyzed using RT-quantitative PCR and/or immunofluorescence. We found 2B11.3 caused glomerular endocapillary hypercellularity in WT mice with glomerular infiltration of larger numbers of CCR2-expressing macrophages and neutrophils phagocyting immune complex, whereas B1 induced wire-loop lesions. In glomerular endocapillary hypercellularity, CCL2 was identified as the ligand involved in the CCR2-positive cell infiltration; it was expressed by glomerular endothelial cells and macrophages. Notably, 2B11.3-induced glomerular endocapillary hypercellularity converted to wire-loop lesions with reduced glomerular macrophage and neutrophil infiltration in CCL2-deficient (Ccl2-/-) mice similarly observed in Ccr2-/- mice. Moreover, this histological conversion was also observed when both glomerular macrophage and neutrophil infiltration were inhibited in anti-Ly6G antibody-treated Ccr5-/- mice but not when only glomerular macrophage infiltration was inhibited in Ccr5-/- mice or when only glomerular neutrophil infiltration was inhibited in anti-Ly6G antibody-treated WT mice. In contrast, B1 injection caused wire-loop lesions in Ccl2-/- and Ccr2-/- mice, as observed in WT mice. Moreover, 2B11.3 induced CCL2 from glomerular endothelial cells to a larger extent than B1 when injected into Ccr2-/- mice. In conclusion, the CCL2-CCR2 axis determines whether glomerular endocapillary hypercellularity or wire-loop lesions develop by regulating glomerular infiltration of phagocytic cells: macrophages and neutrophils. © 2024 The Pathological Society of Great Britain and Ireland.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

IMMU-09. IDENTIFICATION OF A NEW THERAPEUTIC ANTIGEN FOR GLIOBLASTOMA USING A MONOCLONAL ANTIBODY LIBRARY FROM PATIENT-DERIVED TUMOR SPHERES

Abstract Glioblastoma (GBM) has a poor prognosis despite intensive treatment by surgery, radiation, and chemotherapy, thus new treatment strategies are urgently needed. Various innovative cancer therapies targeting cancer-specific cell surface antigens, such as chimeric antigen receptor T cell therapy, are developed and more cell surface antigens that is highly specific for GBM cells are needed. Although extensive transcriptome analyses or proteomic analysis of GBM cells were performed, few transcripts specific for GBM cells have been identified. However, GBM cell-specific antigen epitopes formed by post-translational modifications of proteins may have been missed by transcriptome or proteomic analysis, and could still be discovered by thoroughly searching for cancer-specific monoclonal antibodies. In this study, we aim to identify GBM-specific antigens using a monoclonal antibody library from patient-derived tumor spheres. Approximately 25,000 monoclonal antibody-producing hybridomas were establishment using Balb/c mice and patient derived tumor spheres. Among them, two antibodies that bind to glioblastomas and not to normal brain cells were selected and we identified the antigen by the expression cloning method. The antigen is a cell adhesion molecule and has been reported to be involved in the progression of solid tumors and is expected to be a tumor-specific antigen. These results indicate that the strategy shown in this study is useful for identifying antigens that are potentially useful as targets for GBM therapy.

The non-ionizing electromagnetic stimulation enhanced antibody production (NESEAP) effect - Discovery and technological applications.

The rise of biological therapeutics in the global pharmaceuticals market has escalated the demand for quality monoclonal antibodies for healthcare and scientific applications. Reducing costs while enhancing production yields without compromising quality are the main challenges to the growth of this industry today. Over the last two decades non-ionizing radiation has been demonstrated to elicit targeted biological responses in a frequency and dose dependent manner. We hypothesize and design a millimeter wave radiation procedure to enhance the yields of antibody-producing hybridoma cell lines. We demonstrate this method enhances the production of IgA and IgG antibodies from MOPC315.BM and U13.6 cells by a factor of 24.05±3.32 and 1.41±0.03 respectively relative to untreated cells. No treatment associated cytotoxicity was observed in either cell line corroborating physiological viability of irradiated cells. Our results demonstrate proof-of-concept of a novel technique to significantly enhance antibody yields from hybridoma cells which could lead to a reduction in antibody production costs. Further studies will focus on scaling up of this technology and employment of non-contact, tuned electromagnetic stimulation of biological systems for targeted responses.

Development of monoclonal antibodies against Rhodococcus equi virulence-associated protein N and their application to pathological diagnosis.

Since 2015, virulence-associated protein N (vapN)-positive Rhodococcus equi has been isolated from pyogenic lesions in ruminants in multiple countries, including Japan, supporting the widespread distribution of this pathogen in ruminants, and suggesting that VapN is responsible for the pathogenicity of this infection. However, no immunological diagnostic method has been established for this infection. In this study, we attempted to produce anti-VapN monoclonal antibodies and apply them to immunostaining methods. Mice were immunized intraperitoneally or subcutaneously with 5 µg of recombinant VapN (rVapN). After cell fusion and cloning by limiting dilution, we generated three anti-VapN antibody-producing hybridomas (4H4, 5C3, and 5G10). Next, VapN-based immunostaining was performed using a mouse model inoculated with rVapN, the JCM94-3 (VapN-producing) strain, or the JID03-27 (low-producing) strain. Positive reactions were observed in necrotic lesions in the liver and spleen, especially in the rVapN-treated and JCM94-3-treated groups. Immunostaining of goats (n = 2) and cattle (n = 3) presumed to be infected with vapN-positive R. equi was also positive in organs carrying necrotic lesions including the lungs, lymph nodes, abomasum, and ribs. The reactivity of immunostaining differed among the antibodies, with the 4H4 antibody exhibiting the best reactivity and allowing detection at low concentrations. However, the reactivity of the other two antibodies was improved by antigen retrieval treatment. Several known diseases cause necrotizing granuloma formation, and the immunological diagnostic method established in this study can be used to distinguish R. equi infection from similar diseases or identify infection in previously missed cases.IMPORTANCERhodococcus equi can cause infection in ruminants, and its pathogenicity is suggested to be associated with VapN. Despite its wide distribution, no immunological diagnostic method has been developed for VapN-producing R. equi. Against this background, we attempted to develop monoclonal antibodies targeting VapN and assess their application in immunostaining. In the study, mice were immunized with recombinant VapN, and cell fusion and cloning by limiting dilution permitted the generation of three antibody-producing hybridomas. The utility of the antibodies produced from the hybridomas in immunostaining was demonstrated using an infected mouse model, and the antibodies were further applied to previously reported cases of R. equi infection in goats and cattle. Although the 4H4 antibody induced the strongest reactions, the reactivity of two other antibodies was improved by antigen retrieval. Our monoclonal antibodies will be utilized to support the definitive diagnosis of suspected R. equi infection, including cases that were previously missed.

A Mouse Monoclonal Antibody Against Human IFN-γ and its Characters

Background and Aims: A monoclonal antibody (mAb) can unambiguously identify, quantify, and purify an antigen or particular epitope at a large scale. The superiority of these antibodies lies in their specificity for the antigenic determinant. So, this study aims to prepare mouse mAb-secreting hybridoma against human gamma interferon (IFN-γ) and determine the produced antibody's characters.
 Materials and Methods: Mouse splenic B lymphocytes immunized with recombinant human IFN-γ were fused with mouse SP2/0 cells. The hybridized cells were selected by hypoxanthine-aminopterin-thymidine and hypoxanthine-thymidine media to obtain monoclonal antibody-producing hybridoma cells. Finally, indirect enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blot were used to confirm the creation of antibody-secreting hybridoma cells.
 Results: mAb against IFN-γ were produced by fusing SP2/0 mouse non-secretory myeloma cell line with the spleen cells of immunized mice. This antibody's indirect ELISA optical density was 2.055 on average, and the desired antibody bands were confirmed in SDS-PAGE compared to Septicol® (commercial antibody). Also, in the western blot, the desired antibody could bind to the antigen. IFN-γ transferred on nitrocellulose membrane. In ELISA and western blot tests, anti-mouse IgG conjugated antibodies were used; therefore, the mAb IgG isotype was taken into consideration.
 Conclusion: In this study, a mouse mAb was obtained by immunization of Balb/C mice and fusion of spleen cells of these mice with the SP2/0 cells, which can specifically bind to recombinant human IFN-γ and can be used to detect IFN-γ secretion in all types of intracellular infections, including latent tuberculosis.

Evaluation the reactivity of a peptide-based monoclonal antibody derived from OmpA with drug resistant pulsotypes of Acinetobacter baumannii as a potential therapeutic approach

BackgroundAcinetobacter baumannii is an opportunistic and antibiotic-resistant pathogen that predominantly causes nosocomial infections. There is urgent need for development nonantibiotic-based treatment strategies. We developed a novel monoclonal antibody (mAb) against a peptide of conserved outer membrane protein A (OmpA) and evaluated its reactivity with different pulsotypes of A. baumannii.MethodsPeptide derived from A. baumannii OmpA was conjugated to keyhole limpet hemocyanin and injected into BALB/c mice. Splenocytes of immunized mice were fused with SP2/0 myeloma cells followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, one monoclone was selected as 3F10-C9 and the antibody was tested for reaction with five different Acinetobacter pulsotypes that were resistant to carbapenem antibiotics. The affinity constant was measured by ELISA. The ELISA, western blotting, indirect immunofluorescence (IFA), and in vitro opsonophagocytosis assays were used to evaluate the reactivity of generated mAb.ResultsThe anti-OmpA antibody reacted with the immunizing peptide and had a high affinity (1.94 × 10−9 M) for its antigen in the ELISA. Specific binding of mAb to OmpA was confirmed in Western blot. IFA assays revealed that mAb recognized specific OmpA on the pulsotypes. Opsonophagocytosis assays showed that the mAb increased the bactericidal activity of macrophage cells. The antibody function was higher in the presence of serum complement.ConclusionsThe peptide-based mAb demonstrated optimal performance in laboratory experiments which may be appropriate in investigation on OmpA in Acinetobacter pathogenesis and development of passive immunization as a novel therapeutic approach.

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis.

Antibodies are central effectors of the adaptive immune response, widespread used therapeutics, but also potentially disease-causing biomolecules. Antibody folding catalysts in the plasma cell are incompletely defined. Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease with increasingly recognized autoimmune features. We found elevated expression of FK506-binding protein 11 (FKBP11) in IPF lungs where FKBP11 specifically localized to antibody-producing plasma cells. Suggesting a general role in plasma cells, plasma cell-specific FKBP11 expression was equally observed in lymphatic tissues, and in vitro B cell to plasma cell differentiation was accompanied by induction of FKBP11 expression. Recombinant human FKBP11 was able to refold IgG antibody in vitro and inhibited by FK506, strongly supporting a function as antibody peptidyl-prolyl cis-trans isomerase. Induction of ER stress in cell lines demonstrated induction of FKBP11 in the context of the unfolded protein response in an X-box-binding protein 1 (XBP1)-dependent manner. While deficiency of FKBP11 increased susceptibility to ER stress-mediated cell death in an alveolar epithelial cell line, FKBP11 knockdown in an antibody-producing hybridoma cell line neither induced cell death nor decreased expression or secretion of IgG antibody. Similarly, antibody secretion by the same hybridoma cell line was not affected by knockdown of the established antibody peptidyl-prolyl isomerase cyclophilin B. The results are consistent with FKBP11 as a novel XBP1-regulated antibody peptidyl-prolyl cis-trans isomerase and indicate significant redundancy in the ER-resident folding machinery of antibody-producing hybridoma cells.

CCR2- and CCR5-mediated macrophage infiltration contributes to glomerular endocapillary hypercellularity in antibody-induced lupus nephritis.

LN comprises various glomerular lesions, including endocapillary hypercellularity with macrophage infiltration. In this study, we aimed to clarify the involvement of macrophage-tropic chemokine receptors in the pathogenesis of these glomerular lesions. MRL/lpr mouse-derived monoclonal IgG3 antibody-producing hybridomas, 2B11.3 and B1, were injected intraperitoneally into BALB/c mice [wild type (WT)] to induce endocapillary hypercellularity and wire-loop lesions, respectively. The expression of chemokine and chemokine receptors was analysed by quantitative real-time PCR and IF. The roles of chemokine receptors in these lesions were evaluated using chemokine receptor-deficient mice or a selective CCR5 antagonist, maraviroc. 2B11.3 caused glomerular endocapillary hypercellularity with a significant number of glomerular CD68-positive macrophages. Further, enhanced expression of CCL2, CCL3, CCR2, CCR5 and CX3CR1 was observed in the renal cortex, compared with B1 injection, which induced wire-loop lesions. In 2B11.3-induced glomerular lesions, CD68 -positive glomerular macrophages expressed CCL2, CCL3, CCR2, CCR5 and CX3CR1, while glomerular endothelial cells expressed CCL2, CCL3, CX3CL1 and CCR2. When 2B11.3 was injected, CCR2-/- and CCR5-/-, but not CX3CR1-/-, mice exhibited reduced endocapillary hypercellularity, attenuated glomerular macrophage infiltration and improved serum blood urea nitrogen levels. Only CCR2-/- mice developed wire-loop lesions. B1 injection caused wire-loop lesions in these chemokine receptor-deficient mice to a similar extent as WT. Maraviroc treatment reduced 2B11.3-induced endocapillary hypercellularity and improved serum blood urea nitrogen levels. CCR2 and CCR5 regulate glomerular macrophage infiltration and contribute to the development of glomerular endocapillary hypercellularity in LN. CCR5 inhibition can be a specific therapy for endocapillary hypercellularity without inducing wire-loop lesions.

A New Workflow to Generate Monoclonal Antibodies against Microorganisms

Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.

In vitro immunization approach to generate specific murine monoclonal IgG antibodies

Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophilain vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species.

Development and application of monoclonal antibody against IgM of hybrid snakehead ( Channa maculata ♀ × Channa argus ♂)

Hybrid snakehead (C. maculate ♀ ×C. argus♂) culture has become an important aquaculture in China. However, severe disease caused by bacterial and viral infections resulted in enormous economic losses in the recent years. The effective disease control strategies have not been developed due to limited understanding of the immunology of this species so far. In this study, monoclonal antibodies (mAb) against serum immunoglobulin M (IgM) purified from hybrid snakehead was developed by stimulating mice, of which spleen cells were fused with myeloma cells to generate stable and effective antibody-producing hybridoma cells. The mAb clone number named as 1C3-1–12 and the mAb was further screened successfully using an indirect enzyme-linked immunosorbent assay (iELISA). Western blot analysis showed that the mAb 1C3-1–12 can specifically react with the heavy chain of IgM. There were no cross-reactions with sera from three other teleosts. Meanwhile, the mAb 1C3-1–12 can effectively detect serum IgM levels of hybrid snakehead and its evolutionally closely related species. Moreover, the mAb 1C3-1–12 was found to exclusively recognize the IgM+ B cells of hybrid snakehead by indirect immunofluorescence assay (IFA). Collectively, we developed a useful and specific monoclonal antibody against serum IgM from hybrid snakehead and will be an important immunological tool for better understanding the function of the immune system of hybrid snakehead. It may lie as a foundation for the establishment of diagnosis of disease and the development of effective vaccine.

Generation and characterization of D-dimer specific monoclonal antibodies for use in latex agglutination test.

The commercially available D-dimer assays used in the clinical practice often show differences in the results, and their specificity and sensitivity are rather unsatisfactory. Our aim was to develop a new monoclonal antibody against D-dimer with a proper specificity, and estimating its suitability using in a latex agglutination diagnostic test. Monoclonal antibodies were generated using hybridoma technology. Their titer was determined by a self-developed ELISA method. The cross-reactions of the antibodies were tested. Characterization of the epitope specificity of a selected antibody was performed through digestion of D-dimer followed by Western blotting. The amino acid sequences of the active antigen fragments were determined. According to the ELISA results, 38 cell groups were constated as antibody-producing hybridomas, among them 7 gave raised titer of antibody and were cloned. Based on the cross-reaction analysis, none of the antibodies gave cross-reaction with fibrin-E and fibrinogen-E fragments but reacted with fibrin D and fibrinogen D fragments. A low cross-reaction was showed with fibrinogen and fibrin X and Y. Contrary to the others, antibody 2B9 gave no cross-reaction with fibrinogen and reacted weakly with fibrin X and Y fragments. According to the epitope analysis the antibody 2B9 binds to amino acids 94–99 and to amino acids 140–147 on the beta chain and it recognizes the amino acids 23–32 and 93–98 on the gamma chain of D-dimer. Considering the characteristics of the above mentioned monoclonal antibody 2B9, we found that it is suitable to be a basis for a D-dimer diagnostic test with proper specificity.

Production and Characterization of Monoclonal and Polyclonal Antibodies Against Truncated Recombinant Dickkopf-1 as a Candidate Biomarker.

Several studies have reported an increased serum level of Dickkopf (DKK-1) protein in a variety of cancers, including multiple myeloma, lung, colorectal, bone loss, and Alzheimer's disease. This protein has potential to be used as a biomarker for the diagnosis of some cancers, especially bone loss in multiple myeloma. In the present study, to measure the concentration level of DKK-1 protein, rabbit polyclonal antibody (pAb) and mouse monoclonal antibodies (mAbs) were produced against this protein. New Zealand white rabbits and BALB/c mice were immunized with the chimeric recombinant DKK-1 antigen. Immunized mouse spleen cells were fused with SP2/0 cells to generate anti-rDKK-1 antibody-producing hybridoma cells. Antibodies were purified by protein A affinity chromatography and assessed using sodium dodecyl sulfate polyacrylamide gel, western blotting and enzyme-linked immunosorbent assay. These results implied that the pAb and mAb were produced against the DKK-1 protein. The Kd value of 5 × 10-9 M was recorded for the mAb MR6F3 toward native DKK-1, and the Ig isotype was identified as IgG2b. No cross-reactivity was shown with DKK-2 by MR6F3. Collectively, our results revealed that the produced pAb and mAb could be used in the measurement of DKK-1 protein.

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies.

The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten-horseradish peroxidase conjugate (hapten-HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP-Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG-Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.

Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in Humans In Vivo.

Oxidatively modified low-density lipoprotein (oLDL) is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA) is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA) and Cu++-oxidized LDL IgG/IgM (oLAb) ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodies in vivo. Furthermore, using these antibodies for in vitro experiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu++-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.

A Novel Fusion Protein, AChR-Fc, Ameliorates Myasthenia Gravis by Neutralizing Antiacetylcholine Receptor Antibodies and Suppressing Acetylcholine Receptor-Reactive B Cells.

Most patients with myasthenia gravis (MG) have elevated levels of autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction, which leads to muscle weakness. We developed a fusion protein, AChR-Fc, as a novel therapeutic biomolecule for patients with MG and examined its efficacy. AChR-Fc was expressed by Chinese hamster ovary cells and purified. We examined the neutralizing activity and cellular cytotoxicity of AChR-Fc using anti-AChR antibody-producing hybridoma cells and serum samples from 16 patients with MG. The effects of AChR-Fc in vivo were also examined using rat MG models. AChR-Fc bound to anti-AChR antibodies and exhibited cytotoxicity against patient-derived antibody-producing B cells. Additionally, a dose-dependent improvement in the clinical signs of disease was observed in a rat MG model. AChR-Fc can diminish signs of MG by neutralizing anti-AChR antibodies and enhancing cytotoxicity against autoantibody-producing B cells. Thus, AChR-Fc can be a novel therapeutic biomolecule for patients with MG.

Antibodies and Selection of Monoclonal Antibodies.

Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

A Dual-Chamber System for Screening Cytotoxic Effects of Hybridoma-Produced Antibodies

There are many methods for evaluating the cytotoxic effect of monoclonal antibodies (MAbs) against cancer cells. Most of these methods require either purified MAbs or biological solutions (e.g., cell culture supernatants, ascitic fluids) containing high concentrations of MAbs. This makes the primary screening of antibody-producing hybridomas for specific cytotoxic antibodies a challenging task. Addressing this issue, this work introduces a high throughput screening method, which enables the identification of cytotoxic antibodies using primary hybridoma populations without prior antibody concentration and/or purification. The method is comprised of a dual-chamber system, where antibody-producing hybridomas and target cancer cells are co-cultured but separated by a porous membrane in which the pore size is sufficient for the diffusion of antibody molecules. The MAbs produced in the system continuously diffuse through the membrane between the two chambers and interact with the target cells placed on the other side of a membrane, resulting in death or proliferation arrest of these cells, if MAbs are cytotoxic or cytostatic. The cytotoxic/cytostatic effect can be registered by measuring the viability of target cells. The advantage of this method is that purification or concentration of antibodies secreted by hybridomas is not required. In addition, this method does not require MAb-secreting hybridomas, which are subcloned or have a high level of MAb production. The method may serve as an effective primary high throughput screening for cytotoxic antibodies.

A Monoclonal Antibody Against Leptin

Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.