The start of an autoimmune disease: idiotypic network during early progression of myasthenia gravis

Anti-acetylcholine-receptor antibodies of IgG and IgM classes and antiidiotypic a antibodies were determined in patients with myasthenia gravis at various times after the start of the disease. Patients with a disease duration of less than one year had a higher prevalence of antiidiotypic antibodies (31/32) than patients who had had the disease for more than 5 years (49/79), and the concentration of antiidiotypic antibodies was also higher in patients with early disease (p < 0.005). The concentrations of antiidiotypic antibodies decreased during progression of the disease concomittant with an increase in IgG anti-receptor antibodies. A change from IgM to IgG anti-receptor antibody production was also found. In two patients, who developed myasthenia after bone-marrow grafting and who were followed before start of disease, antiidiotypic antibodies appeared before anti-receptor antibodies and before symptoms of myasthenia were present.The high prevalence and concentration of antiidiotypic antibodies in early disease indicate that development and expression of antiidiotypic antibodies are important in early myasthenia gravis.     

Isoelectric focusing of cross-linked monoclonal antibody heterodimers, homodimers and derivatized monoclonal antibodies

Anti-tumor monoclonal antibodies were cross-linked to the anti-CD3 T-cell antibody OKT3 by the use of the heterobifunctional cross-linker succinimidyl-3-(2-pyridyldithio)propionate. Derivatized monoclonal antibodies, heterodimers, and homodimers were resolved by analytical isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Isoelectric points of the derivatized antibodies were lower than native antibodies, consistent with lysine derivatization. Antibodies derivatized with 2-iminothiolane were equivalent or slightly higher in pI compared with native antibodies. Heterodimers focused in microheterogeneous bands between the pI extremes of the parent derivatized antibodies. The isoelectric points of homodimers were lower than those of parental antibodies and could be distinguished from heterodimers. Reduced and alkylated heterodimers were resolved into their constituent antibodies by isoelectric focusing.     

Recovery of the contact site A glycoprotein of Dictyostelium discoideum from sodium dodecyl sulfate-polyacrylamide gel and characteristics of monoclonal antibodies against the recovered protein

Monoclonal antibodies were produced against a cell-cell adhesion (contact site A) glycoprotein of Dictyostelium discoideum, isolated by preparative gel electrophoresis. The glycoprotein was recovered by electroelution from a polyacrylamide gel strip and used for the production of monoclonal antibodies. Four of the five antibodies obtained bound specifically to the protein moiety of the contact site A glycoprotein. The specificities of the antibodies were in striking contrast to those of antibodies raised against the contact site A glycoprotein purified by Triton X-114 phase separation and DEAE chromatography. The majority of the latter antibodies recognized the carbohydrate moiety of the contact site A glycoprotein and cross-reacted heavily with other membrane glycoproteins.     

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods

Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.     

Docking of Antibodies into the Cavities of DNA Origami Structures

Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme‐linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation‐selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)‐modified strands are inserted into the cavity to form NTA–metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.     

Hydrophobic interaction chromatography at low salt concentration for the capture of monoclonal antibodies

We evaluated hydrophobic interaction chromatography (HIC) at low salt concentration for the capture of proteins from feed stocks by using monoclonal antibodies as model samples. It was indicated that the HIC at low salt concentration on critical hydrophobicity supports has a potential for capturing hydrophobic monoclonal antibodies directly from large volumes of feed stocks and recovering bound monoclonal antibodies in high yield. On the other hand, the HIC at low salt concentration did not seem so useful for the capture of weakly hydrophobic monoclonal antibodies. The recovery of weakly hydrophobic monoclonal antibodies from columns packed with critical hydrophobicity supports was not quantitative and significantly decreased as the residence time of the monoclonal antibodies in the columns became longer.     

Site-directed photochemical disruption of the actin cytoskeleton by actin-binding Rose Bengal-conjugates

The in situ light-induced, non-enzymatic digestion of cytoskeletal actin by a xanthene dye conjugated to heavy meromyosin, anti-actin antibodies and/or anti-myosin antibodies is reported. The dye Rose Bengal was conjugated to either anti-actin antibodies, anti-myosin antibodies or heavy meromyosin. Under our experimental conditions, visible light induced the non-enzymatic breakdown of cytoskeletal actin when mammalian tissue culture cells were probed either with Rose Bengal-conjugated anti-actin and/or anti-myosin antibodies. Similar results were obtained when tissue culture cells were probed with Rose Bengal-conjugated heavy meromyosin before irradiation with visible light. The in situ photochemical reaction depended on the presence of actin-binding Rose Bengal-conjugates.     

Tandem purification of mouse IgM monoclonal antibodies produced in vitro using anion-exchange and gel fast protein liquid chromatography

A tandem chromatographic procedure was used to isolate rapidly mouse IgM monoclonal antibodies produced by cultivation of hybridomas in vitro. Hybridoma culture supernatants containing mouse IgM monoclonal antibodies were first chromatographed on an anion-exchange Mono Q column connected to a fast protein liquid chromatography system. This anion-exchange step offers the advantage of obtaining IgM antibodies in a concentrated form. The IgM-rich fractions from the Mono Q column were then injected on a gel filtration Superose 6 column equilibrated with a low-ionic strength buffer and eluted with a high-ionic strength buffer. Assessment of the purity of isolated IgM monoclonal antibodies was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis together with a Coomassie Brillant Blue R 250 staining technique. Assessment of the immunoreactivity of isolated IgM monoclonal antibodies was performed by an enzyme linked immunosorbent assay using a solid phase adsorbed antigen against which IgM monoclonal antibodies were directed. The chromatographic procedures described allows the rapid isolation of mouse IgM monoclonal antibodies produced in vitro at a high degree of purity and in an immunoreactive state.     

Abzyme generation using an anti-idiotypic antibody as the “internal image” of an enzyme active site

Since the two reports published in 1986 by the laboratories of R. Lerner and P. G. Schultz, it has been clearly established that antibodies may be induced to act as catalysts in numerous chemical reactions. In all cases, catalytic antibodies were elicited using a substratebased approach. In the present article, we propose an alternative and complementary enzyme-based approach to generate catalytic antibodies. This approach uses the properties of anti-idiotypic antibodies to generate internal images of enzyme active sites. Experimental results are discussed for polyclonal and monoclonal anti-idiotypic antibodies.     

One target, different effects: a comparison of distinct therapeutic antibodies against the same targets

To date, more than 30 antibodies have been approved worldwide for therapeutic use. While the monoclonal antibody market is rapidly growing, the clinical use of therapeutic antibodies is mostly limited to treatment of cancers and immunological disorders. Moreover, antibodies against only five targets (TNF-α, HER2, CD20, EGFR, and VEGF) account for more than 80 percent of the worldwide market of therapeutic antibodies. The shortage of novel, clinically proven targets has resulted in the development of many distinct therapeutic antibodies against a small number of proven targets, based on the premise that different antibody molecules against the same target antigen have distinct biological and clinical effects from one another. For example, four antibodies against TNF-α have been approved by the FDA -- infliximab, adalimumab, golimumab, and certolizumab pegol -- with many more in clinical and preclinical development. The situation is similar for HER2, CD20, EGFR, and VEGF, each having one or more approved antibodies and many more under development. This review discusses the different binding characteristics, mechanisms of action, and biological and clinical activities of multiple monoclonal antibodies against TNF-α, HER-2, CD20, and EGFR and provides insights into the development of therapeutic antibodies.     

Induction of antibodies to pseudorabies virus by immunization with antiidiotypic antibodies

We have induced the expression of high titres of antibodies to pseudorabies virus (PRV) by immunization of A/J mice with polyclonal antiidiotypic antibodies directed against each of 2 BALB/c monoclonal, neutralizing anti-PRV antibodies. The anti-PRV antibodies induced by one of the antiidiotypic antibody preparations had an affinity for PRV comparable to that of the original monoclonal antibody. Mice immunized with antiidiotypic antibodies were protected to a significant degree against viral infection but the degree of protection was lower than that conferred by passive transfer of monoclonal neutralizing antibody or by immunization with attenuated virus.     

毛细管电泳技术在单克隆抗体药物分析中的应用

单克隆抗体药物在生物制药行业占有重要地位,是生物医药领域发展的主要方向。因此,单克隆抗体药物的质量控制已成为全球生物制药企业及法规机构关注的热点,对单克隆抗体药物精确表征的需求日益增加。毛细管电泳技术具有分离效率高、分析速度快、分离模式多、样品用量少等特点,已成为单克隆抗体药物分析和质量控制的重要手段。该文对毛细管凝胶电泳、毛细管等电聚焦、毛细管区带电泳等模式在单克隆抗体药物的纯度分析、等电点测定、电荷异质性分析和N-寡糖分析的应用进行综述,以期为国内单克隆抗体研究开发和生产的企事业单位提供技术参考。     

Rapid monitoring of high-mannose glycans during cell culture process of therapeutic monoclonal antibodies using lectin affinity chromatography

We developed a simple high-performance liquid chromatography assay to monitor high-mannose glycans in monoclonal antibodies by monitoring terminal alpha-mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2-aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has a strong affinity to alpha-mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase-treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal-mannose binding to the column and comparable data trending with high mannose levels by 2-aminobenzamide assay confirmed that terminal-mannose levels measured by the Concanavalin A column can be used as a surrogate for the prediction of high-mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for the prediction of high-mannose content in samples compared with traditional glycan profiling by 2-aminobenzamide or mass spectrometry-based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two-dimensional analysis, high-mannose data could be relayed to the manufacturing team in less than 30 min, allowing near-real-time monitoring of high-mannose levels in the cell culture process.     

Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.     

A FILTER BINDING ASSAY FOR CHARACTERIZATION OF ANTIBODIES TOWARDS DAMAGED DNA

A nitrocellulose filter binding assay was used to characterize rabbit antibodies specific for UV-irradiated DNA. The dissociation constant for the UV-DNA-antibody complex was found to be 4.2 × 10⁻¹² M. No significant binding to unirradiated DNA was observed. Unlabelled single-stranded and double-stranded DNA competed with equal efficiency for the radioactively labelled antigen (UV-irradiated φX174 DNA). The antibodies also bound to OsO₄-treated DNA, suggesting that these polyclonal antibodies also recognize minor photoproducts. Caffeine efficiently decreased binding of the antibodies to UV-irradiated DNA.     

Microplate-based screening methods for the efficient development of sandwich immunoassays

The selection of suitable antibodies is a critical step in immunoassay development, since the final assay performance is predetermined by this decision to a large extent. Particularly, the screening for matching pairs in sandwich immunoassays is difficult, if both antibodies are derived from one species or when monoclonal antibodies are only available as cell supernatants. Several microplate-based approaches for in situ labeling of detection antibodies were tested, in order to avoid time consuming purification of antibodies for enzyme conjugate synthesis. We investigated labeling with anti-species antibodies and Fab fragments thereof, labeling with protein G and biotinylation of cell supernatants without prior purification. Antibodies against peanut proteins were used as a model and signal-to-blank ratios were used in all cases as a measure of the antibody pair performance. Amongst the investigated approaches, preincubation of the detection antibody with labeled anti-species antibody turned out to be most suitable under our conditions. Diagrams, showing the performance of all possible antibody combinations, were generated with this method and were compared to results obtained with covalently labeled detection antibodies. Finally, a flowchart is presented, suggesting an efficient strategy for the development of highly sensitive sandwich immunoassays.     

Directed antibody-engineering techniques and their applications in food immunoassays

The use of antibodies in immunodiagnostics has achieved new insights with recombinant technologies. This review summarizes the methods used to produce recombinant antibodies and those to tailor their properties. Finally, we address the advantages and the possibilities of recombinant antibodies in immunoanalytical applications through examples with the main focus on applications related to food quality and safety analysis.     

Antibody-scanning and epitope-tagging methods; molecular mapping of proteins using antibodies

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.     

Superantibodies

The antibody molecule possesses a number of so-called unconventional binding sites in the variable domain that are expressed and function independently from the antigen-binding site. These sites are encoded in the germiline, predominantly in framework residues. By this definition, these sites function as part of the innate immunity, and are not subject to antigendriven mutation and maturation. In this article, we focus on the evidence for the function and utility of the self-binding domain. The self-binding or autophilic domain has been discovered on murine germline-encoded antibodies from the S107/T15 Vh family. Autophilic antibodies form self-complexes after attaching to targets, but remain monomeric in solution. A peptide has been identified that confers self-binding if chemically attached to antibodies. Because this modification enhances the overall avidity of antibodies for target binding, therapeutic and diagnostic antibodies can be biotechnologically improved. The concept of super antibodies is introduced here to describe the unique coexistence and synergism of acquired immunity with innate immunity via antigen-specific and unconventional functional domains. As not every antibody qualifies as a super antibody, biotechnology engineering can produce superantibodies with superior targeting and therapeutic properties.