Rela Koppel

EFRH-phage immunization of Alzheimer's disease animal model improves behavioral performance in Morris water maze trials.

We have developed an immunization procedure for the production of effective anti-β-amyloid (anti-Aβ) antibodies, using filamentous phage displaying only 4 amino acids. The EFRH sequence, encompassing amino acids 3–6 of the 42 residues of Aβ peptide, was found previously to be the main regulatory site for amyloid modulation and the epitope of anti-aggregating antibodies. Engineered filamentous phage enable the display of various numbers of EFRH copies on the phage and serve as potent carriers of antigens. In the present study we have found that phage displaying high EFRH copy number are effective in eliciting humoral response against the EFRH sequence, which in turn relieves the amyloid burden in the brains of amyloid precursor protein Tg mice and improves their ability to perform cognitive tasks.

Immunostaining of calmodulin and aluminium in Alzheimer's disease-affected brains

Previous in vitro studies have shown that Al(3+) binds to calmodulin, inducing alterations in its capability to interact with target proteins, accompanied by loss of immunological recognition by its conformational specific monoclonal antibody CAM1. In spite of the wealth of data of calmodulin action in vitro, little information is available on the possible involvement of this protein in the pathology typical of Alzheimers disease. In the present study, we investigated calmodulin immunoreactivity in post-mortem human brains affected by Alzheimers disease, compared with age-matched control brains. Conformational monoclonal antibodies raised against Ca(2+)-calmodulin, namely CAM1 and CAM4, were used in this study for the characterization of calmodulin. Calmodulin immunorecognition by monoclonal antibody CAM1 was found to be lost in cortical tissue sample from brains affected by Alzheimers disease. This finding leads to the hypothesis of a new, possibly inactive, conformation of the molecule during the disease. On the other hand, CAM4 immunoreactivity was decreased in neurons of brains affected by Alzheimers disease. Anti-Al(3+) monoclonal antibodies revealed instead more marked aluminium immunoreactivity in the affected brains compared to normal ones. The loss of CAM1 immunoreactivity and the occurrence of large amounts of aluminium suggest an alteration of the active conformation of calmodulin in disease-affected brains. These alterations could be involved in the development of Alzheimers disease pathology.

Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes.

Site-specific modification of monoclonal antibodies was achieved by oxidation of the carbohydrate moieties of antibodies which are located remote from the antigen binding sites. Sialic acid and galactose are terminal sugars of these carbohydrate chains. Concomitant treatment of the antibodies with neuraminidase and galactose oxidase generated aldehyde groups in the oligosaccharide moieties of immunoglobulins which reacted selectively with amino or hydrazide groups of the matrix. Subsequent immobilization of neuraminidase and galactose oxidase on Eupergit C-adipic dihydrazide proved to be an efficient and selective system for the enzymic oxidation of the monoclonal antibodies without impairing their immunological activity. Oriented immobilization of enzymically oxidized monoclonal antibodies on hydrazide or amino Eupergit C derivatives thus leads to the formation of antibody matrix conjugates which possess high antigen-binding activities.

Use of monoclonal antibodies for the preparation of highly active immobilized enzymes.

Publisher Summary This chapter describes a novel method for the preparation of highly active immobilized enzymes. The method is based on the binding of enzymes to suitable carriers through monoclonal antibodies that bind to the enzyme with high affinity without affecting its catalytic activity. The cell fusion technique developed by Kohler and Milstein makes it possible to prepare a great variety of monoclonal antibodies directed toward specific, well-defined antigenic sites of a protein. The available data suggest that for many enzymes it might be possible to prepare corresponding monoclonal antibodies that bind to them with high affinity without affecting their catalyic activity. Binding of such antibodies to insoluble carriers should thus yield carrier-monoclonal antibody conjugates that bind readily with the corresponding enzymes to yield highly active immobilized enzymes. In such immobilized enzyme preparations, the enzyme is bound through a specific, preselected, well-defined site of the enzyme.

Activity of monoclonal antibodies in prevention of in vitro aggregation of their antigens

The availability of monoclonal antibodies (mAbs) which bind to a specific antigen at distinct and well-defined sites led to a better understanding of the effect of highly specific antigen-antibody interactions on antigen behavior. We found that appropriate mAbs interact with their antigens at sites where unfolding is started, leading to stabilization, refolding and suppression of further aggregation. These effects were found to be related to the localization of the antigen site of the antibody. In the following we study the interaction of carboxypeptidase A (CPA) and Alzheimer’s β-amyloid peptide (βAP) with some of their selected monoclonal antibodies. From a large panel of mAbs we selected two mAbs which exhibit significant chaperone-like activity in the refolding and prevention of CPA aggregation induced by heat and excess of Zn ions. CPA exposed to aggregation conditions maintains its solubility and catalytic properties in the presence of mAbs CP10 and CP9. In vitro aggregation of βAP [1-40] was inhibited by the presence of one of its mAbs, called AMY-33, but not by mAb 6F/3D, reinforcing the dependence of prevention of aggregation on the localization of aggregating epitopes. Identification of sequences or regions that may play a role in the protein folding pathway via appropriate mAbs may lead to understanding and prevention of protein aggregation.

Enhanced activity of immobilized dimethylmaleic anhydride-protected poly- and monoclonal antibodies

The effect of reversible protection of the free amino groups of poly- and monoclonal antibodies by dimethylmaleic anhydride on their binding activity following immobilization onto various carriers was studied. The treatment with dimethylmaleic anhydride resulted in a 1.6-1.8-fold increase in the activity of immobilized goat anti-mouse immunoglobulin antibody immobilized onto different epoxy containing carriers and a 3-10.7-fold increase in the activity of immobilized monoclonal antibodies specific for carboxypeptidase A. The increase in activity was most pronounced at low antigen to carrier loads and over a wide range of modifier to protein ratios. The application of reversible protection of antibodies may permit the development of highly active immobilized antibody preparations for use in immunoaffinity purification.

IgM detection via selective recognition by mannose-binding protein

Ca(2+)-dependent mannose-binding proteins (MBPs) belong to the family of animal lectins isolated from the liver and serum of rabbits, humans and rodents. They perform in vivo as defense molecules that act as opsonins by enhancing the clearance of mannose-rich pathogens and have been used in vitro for the purification of immunoglobulin M (IgM). In this study, we used MBPs as a sensitive and specific reagent for the detection of IgM due to their high specificity for mannose found only in IgM carbohydrate regions. MBP performed as a sensitive alternative to the usually used anti-IgM, where very low concentrations of IgM should be detected. IgM plays a central role in the initial response of the immune system to the invasion of foreign pathogens, as the early detection of the appearance of pathogenic IgM in biological fluids is of great significance in the diagnosis and treatment of many acute pathological cases. The development of a highly sensitive and reliable assay for the detection of low concentrations of IgM based on covalent binding on epoxy film-coated surfaces and selective recognition of IgM by MBP may be of clinical importance.

Highly active enzyme preparations immobilized via matrix-conjugated anti-Fc antibodies

Abstract A novel method for enzyme immobilization to achieve highly active enzyme preparations is described. The enzyme is first reacted in solution with a specific monoclonal antibody (mAb) which does not interfere with its enzymic activity and which possesses a high affinity towards the enzyme, The immunocomplex is then reacted with immobilized anti-Fc antibodies which are coupled to the matrix by methods that preserve maximum immunological activity. Horseradish peroxidase (HRP) immobilization on epoxy-activated beads (CB6200) was chosen as a model system for this approach. Coupling of HRP via its mAbs to anti-Fc antibodies immobilized yielded the best results, corresponding to binding of 325 ng of fully active enzyme per bead. Binding of the enzyme to anti-HRP mAb directly immobilized on the beads was considerably lower.

Affinity purification of a mannose-binding protein, a sensitive tool in the diagnostics of IgM, via site-directed phosphorylated mannan bound to alumina

Ca2+ -dependent mannose-binding proteins (MBPs) belong to the family of animal lectins. They perform in vivo as defence molecules that act as opsonins by enhancing the clearance of mannose rich pathogens and have been used in vitro for the purification of IgM. MBPs have been previously isolated by methods based on binding the protein moiety of various mannan species to different matrices. However, the mannan-protein complexes did not have a constant protein content and the yield of the isolated MBPs was variable. In the present study we describe a new approach for the affinity purification of MBPs based on the main polysaccharide moiety of the complex. After removal of residual phosphate groups naturally occurring at the C-3 position of the sugar, which interfere with MBP recognition, the mannan was phosphorylated enzymatically at C-6, at which position the OH group is not required for lectin binding. The enzymatically phosphorylated mannan bound to an alumina column was used successfully for MBP separation from rabbit serum. The mannose-binding protein obtained was used in our study for diagnostic purposes in the identification and determination of very low concentrations of IgM.

Enzyme Immobilization via Monoclonal Antibodies

A novel method for the preparation of highly active immobilized enzymes is described. The method is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The cell-fusion technique developed by Kohler and Milstein’ makes it possible to prepare a great variety of monoclonal antibodies directed towards specific, well-defined antigenic sites of a protein. The available data2 suggest that for many enzymes it might be possible to prepare corresponding monoclonal antibodies that bind to them with high affinity (>lo* M-I) without affecting their catalytic activity. The binding of such antibodies to insoluble carriers should thus yield carrier-monoclonal antibody conjugates, which bind readily with the corresponding enzymes to yield highly active immobilized enzymes. In such immobilized enzyme preparations the enzyme is bound via a specific, preselected, well-defined site of the enzyme. It should be noted that the monoclonal antibody component of these immobilized enzyme preparations represents a rather extended spacer separating the enzyme from the carrier. One might, therefore, expect that enzymes immobilized via their corresponding monoclonal antibodies would undergo less steric interference by the carrier and, consequently, have more freedom to react even with higher molecular weight substrates. The applicability of the method has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations, as follows: a mouse monoclonal antibody (mAb 100) to CPA that binds to the enzyme with a high affinity constant without affecting its catalytic activity was prepared, purified, and ~haracter ized.~ Covalent binding of monoclonal antibody to Eupergit C (EC)4 (FIG. l), or noncovalent binding to Sepharose-Protein A (SPA),’ yielded the conjugated carriers EC-mAb and SPA mAb respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb. CPA and SPA-mAb-CPA.‘ Although one can prepare monoclonal antibodies that bind to a corresponding protein antigen (enzyme) with very high binding constants (> 10” M-I), it is obvious that systems a t equilibrium, in which antigen and antibody in solution or suspension are present will contain some free antigen (enzyme). The concentration of the latter can, however, be diminished markedly by increasing the concentration of soluble or immobilized antibody. It should be mentioned that the binding constant describing an